Methods for treating cancer using an immunotoxin

ABSTRACT

The present invention relates to methods for preventing or treating head and neck squamous cell cancer and bladder cancer using an immunotoxin comprising (a) a ligand that binds to a protein on the cancer cell attached to; (b) a toxin that is cytotoxic to the cancer cell. In a specific embodiment, the invention is directed to the prevention or treatment of head and neck squamous cell cancer or bladder cancer using Vb4-845, which is a recombinant immunotoxin comprising a humanized, MOC31-derived, single-chain antibody fragment that is fused to a truncated form of  Pseudomonas  exotoxin A. Also encompassed by the invention are combination therapy methods, including the use of reduced dosages of chemotherapeutic agents, for the prevention or treatment of cancer. Also encompassed by the invention are formulations and methods for direct administration of the recombinant immunotoxin to the carcinoma, for the prevention or treatment of cancer.

RELATED APPLICATIONS

This continuation application claims the benefit of and priority to U.S.patent application Ser. No. 12/698,434 filed Feb. 2, 2010, which is acontinuation of U.S. application Ser. No. 10/554,788 filed Nov. 13,2006, which is a national phase of PCT/CA2004/000637 filed Apr. 30,2004, which claims the benefit of U.S. Provisional Patent ApplicationSer. No. 60/466,608 filed Apr. 30, 2003.

FIELD OF THE INVENTION

The present invention is directed to methods for the prevention ortreatment of cancer by administering to patients having cancer, or atrisk of having cancer, an immunotoxin which binds to an antigenselectively expressed on the surface of cancer cells.

BACKGROUND OF THE INVENTION

Recently, immunotherapy has emerged as a potentially effective newapproach to combat cancer, Murine and humanized/chimeric antibodies, andtheir respective antibody fragments, directed against tumor-associatedantigens (“TAAs”) have been used for diagnosis and therapy of certainhuman cancers.⁵⁻¹³ Unconjugated, toxin-conjugated, and radiolabeledforms of these antibodies have been used in such therapies.

One tumor associated antigen of interest for immunotherapy is Ep-CAM(for Epithelial Cell Adhesion Molecule, which also known as 17-1A, KSA,EGP-2 and GA733-2). Ep-CAM is a transmembrane protein that is highlyexpressed in many solid tumors, including carcinomas of the lung,breast, ovary, colorectum, and squamous cell carcinoma of the head andneck, but weakly expressed in most normal epithelial tissues. The roleof Ep-CAM in cancer formation remains unclear; however, its expressioncorrelates with the rate of cellular proliferation. Ep-CAM-specificantibodies have been used to image and detect primary tumors andmetastases in patients with small cell lung cancer and non-small celllung cancer. Among anti-Ep-CAM MAbs, PANOREX®, which is a murinemonoclonal antibody also known as edrecolomab, had been approved for thetreatment of colon cancer in Germany, and is in clinical trials in theUnited States.¹⁴⁻¹⁵ Of note, however, PANOREX® treatment has beenassociated with undesirable side effects, including abdominal cramps,nausea, transient diarrhea and cutaneous urticariallesions.^(39, 41, 51) Clinical trials with other Ep-CAM-targetedantibodies have been less successful; antibody 30 BIS-1 was associatedwith peripheral vasoconstriction, dyspnea and fever, and antibody3622W94 was associated with acute necrotizing pancreatitis.^(39-41, 57)The search for an effective, low-toxicity, anti-Ep-CAM antibodycontinues: a fully humanized anti-Ep-CAM antibody, MT201, purported toact via Antibody-Dependent Cellular Cytotoxicity (“ADCC”), has beenreported.⁵⁸ A humanized, stabilized, single-chain, anti-Ep-CAM antibody,4D5MOC-B, which is derived from murine monoclonal antibody MOC31, hasalso been developed, and is described in International PatentApplication No. PCT/EP00/03176, Publication No. WO 00/61635, filed Apr.10, 2000 and published Oct. 19, 2000, and in Willuda et al.⁵⁹ Thesepublications do not disclose the use of the humanized antibody in thetreatment of head and neck squamous cell carcinoma (HNSCC) or bladdercancer.

As stated above, one of the cancers associated with increased expressionof Ep-CAM is squamous cell carcinoma of the head and neck (“HNSCC”).Ep-CAM expression correlates with the progression of squamous cellcarcinoma of the head and neck in humans. HNSCC is presently the sixthmost common cancer in the world. HNSCC is a disease that causessignificant morbidity, especially with respect to speech and swallowingfunctions. Surgery, radiation therapy, chemotherapy, or combinations ofthese are generally available as treatment options.

Despite all attempts to cure patients afflicted with HNSCC, recurrenceremains the most common cause of failure (in 40%-50% of patients) afterhead and neck cancer therapy. Salvage therapy consists of the sametreatment options as for first line therapy. However, palliative surgeryis often difficult and disfiguring. Furthermore, radiation therapy israrely feasible or beneficial, and chemotherapy does not substantiallyimprove survival rates in HNSCC patients. Prognosis for these patientsremains poor, such that the median survival after recurrence is onlyapproximately six months.

Due to the poor prognosis for HNSCC patients, the impact of the diseaseon quality of life, and the limited treatment options, there isconsiderable interest in, and a compelling need for, the development ofnew tumor-specific therapies, particularly directed to HNSCC.

Bladder cancer is the 7th most common cancer worldwide that results inan estimated 260,000 new cases each year. In Europe, this disease is thecause of death for approximately 50,000 people each year. Carcinomas inthe bladder tissue occur almost entirely within the transitionalepithelium, the surface layer of tissue that lines the bladder, astransitional cell carcinomas. At initial diagnosis, 70 to 90% ofpatients with bladder cancers have superficial disease which involvescarcinomas in the superficial urothelial layer that are noninvasive andexhibit papillary (finger-like projections) tumors. Current treatmentincludes the intravesicular delivery of chemotherapy and immunotherapywith the bacille Calmette-Guerin (BCG) vaccine that involves theadditional risk of systemic infection with the tuberculosis bacterium.Despite this aggressive treatment regime, 70% of these superficialpapillary tumors will recur over a prolonged clinical course, causingsignificant morbidity; approximately 4 to 8% will progress to invasivecarcinomas.

In response to this medical need, there is considerable need in thedevelopment of new, tumor-specific therapies. One novel approach istargeted therapy using an immunotoxin: an antibody conjugated with atoxin. The antibody binds specifically to tumor cells to deliver thetoxin for efficient tumor cell-killing.

SUMMARY OF THE INVENTION

The present invention relates to novel methods for treating head andneck squamous cell carcinoma and bladder cancer by administering, to apatient in need of such treatment, an effective amount of a recombinantimmunotoxin that specifically binds to (and therefore is “targeted to”)a protein on the surface of the cancer cells. Where desired, theimmunotoxin may be co-administered, concurrently administered, and/orsequentially administered with one or more other anti-cancer agents,and/or in conjunction with radiation or surgery.

The invention also relates to methods for preventing, preventingrecurrence, or reducing the rate of recurrence, of a cancer, comprisingdirectly administering an effective amount of an immunotoxin to a siteof suspected occurrence or recurrence.

The invention also relates to methods for reducing the risk ofpost-surgical complications comprising administering directly to thesurgical site an effective amount of an immunotoxin before, during,and/or after surgery for cancer.

The invention also relates to methods for sensitizing a tumor or cancerto another cancer therapeutic comprising administering an effectiveamount of an immunotoxin. The other cancer therapeutic may beadministered prior to, 30 overlapping with, concurrently, and/or afteradministration of the immunotoxin.

The immunotoxin used in the therapeutic methods of the inventioncomprises (a) a ligand that binds to a protein on the cancer cellattached to; (b) a toxin that is cytotoxic to the cancer cell. Thecancer cell binding portion (a) may be linked to the toxin portion (b)by, for example, chemical linking or genetic linking.

In particular, non-limiting embodiments, the ligand binds Ep-CAM. In aspecific, non-limiting embodiment, the ligand is an antibody or antibodyfragment.

Other features and advantages of the present invention will becomeapparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples while indicating preferred embodiments of the invention aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE FIGURES

The invention will now be described in relation to the drawings inwhich:

FIG. 1 is a schematic showing a template for the intratumoraladministration of immunotoxin and/or other cancer therapeutic to a tumormass

FIG. 2. (A) Map of VB4-845. The map depicts the organization of theimmunotoxin's linked 4D5MOCB scFv and ETA252-608 portions, as well asthe various domains, including the histidine tags, PelB signal, linkerregions, the Vl and Vh regions, ETA regions II, Ib, and III, and the ERretention signal. (B) Predictive Three-Dimensional Model of 4D5MOCB-ETA.The structure of the scFv (VL and VH), ETA252-608 (domains II, Ib, andIII), the linking peptide, and both histidine tags are shown.

FIGS. 3A-D and SEQ ID NOS:1 and 2 show the DNA and Amino Acid Sequencesof VB4-845. The nucleotide and polypeptide sequences can be divided intodomains including: the signal sequence for periplasmic expression,histidine tags, CDR 1, 2 and 3 domains, VL domain, VH domain, linkers,ETA domains II, Ib, III, and an ER retention signal KDEL.

FIG. 4. Antitumor Effect of VB4-845 on Human Tumor Xenografts53. Athymicmice bearing Ep-CAM positive tumor xenografts (HT29, SW2, CAL27), or anegative control (COLO320 (o)) were treated i.v. every second day withVB4-845 at 5 μg (9 doses (▪)) or 10 μg (3 doses (▴)). Tumor size isgiven relative to the initial median tumor size of 160 mm3.

FIG. 5. Peritumoral Treatment of Athymic Mice Bearing CAL27 TumorXenografts. Athymic mice bearing Ep-CAM-positive CAL27 tumor xenograftswere treated peritamorally every second day (Mon/Wed/Fri) with VB4-845at 5 μg (9 doses). Tumor size is given relative to the initial mediantumor size.

FIG. 6. Liver Function Upon Treatment With VB4-845 (4D5MOCB-ETA). Forcomparison, the transaminase activity of mice treated with a singlelethal dose of wild-type ETA (85 μg/kg), as described by Schümann etal.⁵⁵⁻⁵⁶, is also shown. Data are expressed as the mean±SD (n=3).

FIG. 7. Histopathological Results in Liver and Spleen Induced byVB4-845. Circle indicates area of necrotic hepatocytes in the 20 μg dosegroup.

DEFINITIONS

As used herein, the term “animal” includes all members of the animalkingdom, including humans. The animal is preferably a human with HNSCCor bladder cancer.

As used herein, the phrase “cancer therapeutic” refers to compounds ortreatments that are effective in treating or preventing cancerincluding, without limitation, chemical agents, otherimmunotherapeutics, cancer vaccines, anti-angiogenic compounds, certaincytokines, certain hormones, gene therapy, radiotherapy, surgery, anddietary therapy.

As used herein, the phrase “effective amount” means an amount effective,at dosages and for periods of time necessary to achieve the desiredresult. Effective amounts of an immunotoxin may vary according tofactors such as the disease state, age, sex, weight of the animal.Dosage regima may be adjusted to provide the optimum therapeuticresponse. For example, several divided doses may be administered dailyor the dose may be proportionally reduced as indicated by the exigenciesof the therapeutic situation.

As used herein, the phrase “humanized antibody or antibody fragment”means that the antibody or fragment comprises human framework regions.The humanization of antibodies from non-human species has been welldescribed in the literature. See for example EP-B1 0 239400 and Carter&Merchant 1997 (Curr Opin Biotechnol 8,449-454, 1997).

As used herein, the phrase “the immunotoxin is administered directly tothe cancer site” refers to direct or substantially direct introductionincluding, without limitation, single or multiple injections of theimmunotoxin directly into the tumor or peritumorally, continuous ordiscontinuous perfusion into the tumor or peritumorally, introduction ofa reservoir into the tumor or peritumorally, introduction of aslow-release apparatus into the tumor or peritumorally, introduction ofa slow-release formulation into the tumor or peritumorally, directapplication onto the tumor, direct injection into an artery thatsubstantially directly feeds the area of the tumor, direct injectioninto a lymphatic vessel that substantially drains into the area of thetumor, direct or substantially direct introduction in a substantiallyenclosed cavity (e.g., pleural cavity) or lumen (e.g., intravesicular).“Peritumoral” is a term that describes a region, within about 10 cm,preferably within 5 cm, more preferably within 1 cm, of what is regardedas the tumor boundary, such as, but not limited to, a palpable tumorborder. “Direct administration” in the context of prevention ofoccurrence or prevention of recurrence is defined as administrationdirectly into a site at risk for development or recurrence of a cancer.

As used herein, the phrase “ligand that binds to a protein on the cancercell” includes any molecule that can selectively target the immunotoxinto the cancer cell by binding to a protein on the cancer cells. Thetargeted protein on the cancer cell is preferably a tumor associatedantigen that is expressed at higher levels on the cancer cell ascompared to normal cells.

As used herein, the term “MOC-31 antibody” means the murine anti-Ep-CAMor anti-EGP-2 antibody that is known in the art and is available fromcommercial sources such as BioGenex, cat no. MU316-UC, ZymedLaboratories Inc., cat. No. 18-0270 or United States Biological, cat no.M4165.

As used herein, the term “4D5MOC-A” means the humanized scFv MOC31antibody that was grafted onto the artificial human consensus frameworkof scFv 4D5 as described in WO 00/61635 which is incorporated herein byreference.

As used herein, the term “4D5MOC-B” means a stable variant of 4D5MOC-Athat was prepared as described in WO 00/61635 which is incorporatedherein by reference.

As used herein, the term “VB4-845” means an immunotoxin that comprisesa) the scFv humanized antibody 4D5MOC-B that is fused to b) a truncatedform of Pseudomonas exotoxin A that consists of amino acids 252-608.

As used herein, the phrase “pharmaceutically acceptable” refers togeneral clinical use and/or approval by a regulatory agency of theFederal or state government, listing in the United States Pharmacopoeia,or general acceptance by those skilled in the relevant art.

As used herein, “physiologic conditions” for antibody binding reflectbut do not necessarily exactly duplicate the conditions in which anEp-CAM-binding polypeptide would encounter an Ep-CAM molecule in vivo.Binding under physiologic conditions should be reasonably predictivethat binding in vivo will occur.

As used herein, the phrase “preventing cancer” refers to prevention ofcancer occurrence. In certain instances, the preventative treatmentreduces the recurrence of the cancer. In other instances, preventativetreatment decreases the risk of a patient from developing a cancer, orinhibits progression of a pre-cancerous state (e.g. acolon polyp) toactual malignancy.

As used herein, the phrase “reduced dose” refers to a dose that is belowthe normally administered and/or recommended dose. The normallyadministered dose of a cancer therapeutic can be found in referencematerials well known in the art such as, for example, the latest editionof the Physician's Desk Reference.

As used herein, the phrase “treating cancer” refers to inhibition ofcancer cell replication, inhibition of cancer spread (metastasis),inhibition of tumor growth, reduction of cancer cell number or tumorgrowth, decrease in the malignant grade of a cancer (e.g., increaseddifferentiation), or improved cancer-related symptoms.

As used herein, the term “variant” refers to any pharmaceuticallyacceptable derivative, analogue, or fragment of an immunotoxin, anantibody or antibody fragment, a toxin (e.g., Pseudomonas toxin), orcancer therapeutic described herein, A variant also encompasses one ormore components of a multimer, multimers comprising an individualcomponent, multimers comprising multiples of an individual component(e.g., multimers of a reference molecule), a chemical breakdown product,and a biological breakdown product. In particular, non-limitingembodiments, an immunotoxin may be a “variant” relative to a referenceimmunotoxin by virtue of alteration(s) in the Ep-CAM-binding portionand/or the toxin portion of the reference immunotoxin. For example, avariant immunotoxin may contain multimers of the antibody portion and/orthe toxin portion. A variant of the toxin portion of the moleculeretains toxicity of at least 10 percent and preferably at least 30percent in a standard assay used to measure toxicity of a preparation ofthe reference toxin.

A variant immunotoxin having a variation of the Ep-CAM-binding portionof the reference immunotoxin competes with the binding of an anti-Ep-CAMreference antibody, under physiologic conditions, by at least 10 percentand preferably at least 30 percent (and see infra). Competition by 10percent means that, in an assay where a saturating concentration ofanti-Ep-CAM reference antibody is bound to Ep-CAM, 10 percent of thesebound reference antibodies is displaced when an equilibrium is reachedwith an equivalent concentration of the variant anti-Ep-CAM immunotoxinbeing tested. As a non-limiting example, competition between antibodies,or between an antibody and an immunotoxin, is measured by (1) bindinglabeled anti-Ep-CAM reference antibody to Ep-CAM on the surface ofcells, or to an Ep-CAM-coated solid substrate, such that virtually allEp-CAM sites are bound by the antibody; (2) contacting theseantibody-antigen complexes with unlabeled test anti-Ep-CAM antibody orunlabeled test immunotoxin; and (3) measuring the amount of labeledantibody displaced from Ep-CAM binding sites, wherein the amount offreed, labeled antibody indicates the amount of competition that hasoccurred.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have shown that an immunotoxin comprising a humanizedantibody fragment that binds to the extracellular domain of human Ep-CAMlinked to Pseudomonas exotoxin A is effective in treating both head andneck squamous cell carcinoma (HNSCC) and bladder cancer. In particular,the inventors have shown that an immunotoxin comprising a single-chainFv recombinant stabilized and humanized antibody fragment to Ep-CAM thathas been fused to a truncated form of Pseudomonas Exotoxin A (ETA) whichlacks the cell binding domain is cytotoxic against both HNSCC andbladder cancer cells. This immunotoxin binds to Ep-CAM expressed on thecancer cells. Once bound, the immunotoxin is internalized and thePseudomonas Exotoxin A blocks the protein synthesis, therein leading tocell death. Importantly, since most normal mucosal cells and fibroblastsdo not widely express Ep-CAM, and therefore cannot internalize theimmunotoxin, they are protected from the killing effect of the exotoxin.

Accordingly, in one embodiment, the present invention provides a methodfor treating or preventing head and neck squamous cell carcinomacomprising administering to an animal in need of such treatment aneffective amount of an immunotoxin comprising: (a) a ligand that bindsto a protein on the cancer cell attached to; (b) a toxin that iscytotoxic to the cancer cells. The present invention also provides a useof an effective amount of an immunotoxin comprising: (a) a ligand thatbinds to a protein on the cancer cell attached to; (b) a toxin that iscytotoxic to the cancer cells to treat or prevent head and neck squamouscell carcinoma. The present invention further provides a use of aneffective amount of an immunotoxin comprising: (a) a ligand that bindsto a protein on the cancer cell attached to; (b) a toxin that iscytotoxic to the cancer cells in the manufacture of a medicament totreat or prevent head and neck squamous cell carcinoma.

In another embodiment, the present invention provides a method fortreating or preventing bladder cancer comprising administering to ananimal in need of such treatment an effective amount of an immunotoxincomprising: (a) a ligand that binds to a protein on the cancer cellattached to; (b) a toxin that is cytotoxic to the cancer cells. Thepresent invention also provides a use of an effective amount of animmunotoxin comprising: (a) a ligand that binds to a protein on thecancer cell attached to; (b) a toxin that is cytotoxic to the cancercells to treat or prevent bladder cancer. The present invention furtherprovides a use of an effective amount of an immunotoxin comprising: (a)a ligand that binds to a protein on the cancer cell attached to; (b) atoxin that is cytotoxic to the cancer cells in the manufacture of amedicament to treat or prevent bladder cancer.

The ligand that binds to a protein on the cancer cell can be anymolecule that can selectively target the immunotoxin to the cancercells. In one embodiment, the ligand binds to a tumor associatedantigen. Examples of proteins that are expressed on HNSCC cells includeIL-4 receptor, the EGF-receptor, the HER21 neu surface protein andEp-CAM. Examples of proteins that are expressed on bladder cancer cellsinclude EGF-receptor, gp54 and Ep-CAM, In a specific embodiment, theligand binds to Ep-CAM.

In a preferred embodiment, the ligand is an antibody or antibodyfragment. Antibody fragments that may be used include Fab, Fab¹,F(ab′)₂, scFv and dsFv fragments from recombinant sources and/orproduced in transgenic animals. The antibody or fragment may be from anyspecies including mice, rats, rabbits, hamsters and humans. Chimericantibody derivatives, i.e., antibody molecules that combine a non-humananimal variable region and a human constant region are also contemplatedwithin the scope of the invention. Chimeric antibody molecules caninclude, for example, humanized antibodies which comprise the antigenbinding domain from an antibody of a mouse, rat, or other species, withhuman constant regions. Conventional methods may be used to makechimeric antibodies. (See, for example, Morrison et al., Proc. Natl.Acad. Sci. U.S.A. 81, 6851 (1985); Takeda et al., Nature 314, 452(1985), Cabilly et al., U.S. Pat. No. 4,816,567; Boss et al., U.S. Pat.No. 4,816,397; Tanaguchi et al., European Patent Publication EP171496;European Patent Publication 0173494, United Kingdom patent GB 2177096B).The preparation of humanized antibodies is described in EP-B 10 239400.Humanized antibodies can also be commercially produced (Scotgen Limited,2 Holly Road, Twickenham, Middlesex, Great Britain.). It is expectedthat chimeric antibodies would be less immunogenic in a human subjectthan the corresponding non-chimeric antibody. The humanized antibodiescan be further stabilized for example as described in WO 00/61635.

Specific antibodies, or antibody fragments, reactive proteins on HNSCCor bladder cancer cells may also be generated by screening expressionlibraries encoding immunoglobulin genes, or portions thereof, expressedin bacteria with peptides produced from the nucleic acid moleculesencoding the proteins. For example, complete Fab fragments, VH regionsand FV regions can be expressed in bacteria using phage expressionlibraries (See for example Ward et al., Nature 341, 544-546: (1989);Huse et al., Science 246, 1275-1281 (1989); and McCafferty et al. Nature348, 552-554 (1990)). Alternatively, a SCID-hu mouse, for example themodel developed by Genpharm, can be used to produce antibodies orfragments thereof.

The ligand portion of the immunotoxin may be immunoglobulin derived,i.e., can be traced to a starting molecule that is an immunoglobulin (orantibody). For example, the ligand may be produced by modification of animmunoglobulin scaffold using standard techniques known in the art. Inanother, non-limiting example, immunoglobulin domains (e.g., variableheavy and/or light chains) may be linked to a non-immunoglobulinscaffold. Further, the ligand may be developed by, without limitation,chemical reaction or genetic design. Accordingly, in a non-limitingexample, an immunotoxin may comprise (1) an immunoglobulin-derivedpolypeptide (e.g., an antibody selected from an antibody library), orvariant thereof, that specifically binds to HNSCC or bladder cancercells, and (2) a toxin or variant thereof. Such immunoglobulinpolypeptide ligands can be re-designed to affect their bindingcharacteristics to a target a tumor associated molecule, or to improvetheir physical characteristics, for example.

The ligand portion of the immunotoxin need not be immunoglobulin based.Accordingly, an immunotoxin may comprise (1) a non-immunoglobulinpolypeptide (e.g., Affibody®), or variant thereof, that specificallybinds to HNSCC or bladder cancer cells, and (2) a toxin or variantthereof. Such non-immunoglobulin polypeptide ligands can be designed tobind to a target tumor associated molecule. Moreover, non-immunoglobulinpolypeptide ligands can be engineered to a desired affinity or avidity,and can be designed to tolerate a variety of physical conditions,including extreme pH ranges and relatively high temperature.

Indeed, for use in a pharmaceutical composition, the design of anon-immunoglobulin polypeptide with a relatively long half-life atphysiological conditions (e.g., 37° C. in the presence of peptidases)can be advantageous. Furthermore, such molecules, or variants thereof,may demonstrate good solubility, small size, proper folding and can beexpressed in readily available, low-cost bacterial systems, and thusmanufactured in commercially reasonable quantities. The ability todesign a non-immunoglobulin polypeptide is within the skill of theordinary artisan. See, e.g., U.S. Pat. Nos. 5,831,012 and 6,534,628 fortechniques generally adaptable to design, manufacture, and selectdesired binding partners.

Examples of epitope-binding polypeptides include, without limitation,ligands comprising a fibronectin type III domain (see, e.g.,International Publication Nos. WO 01/64942, WO 00/34784, WO 02/32925).Protein A-based affinity libraries have also been used to identifyepitope-binding polypeptides (see, e.g., U.S. Pat. Nos. 5,831,012 and6,534,628) and such libraries may be useful in accordance with thepresent invention to select polypeptides that selectively bind to HNSCCor bladder cancer cells.

Other types of binding molecules are known in the art including, withoutlimitation, binding molecules based on assembly of repeat proteindomains (see, e.g., Forrer et al., 2003, “A novel strategy to designbinding molecules harnessing the modular nature of repeat proteins.”FEBS Lett. 539:2-6; Kohl et al., 2003, “Designed to be stable: crystalstructure of a consensus ankyrin repeat protein.” Proc Natl Acad SciUSA. 100:1700-1705). Libraries of randomly assembled repeat domains maybe useful in accordance with the present invention to select ligandsthat selectively bind to HNSCC or bladder cancer cells.

Several non-immunoglobulin based, epitope-binding polypeptides andmethods for making and using such polypeptides are known in the art(see, e.g., Eklund et al., 2002, “Anti-idiotype protein domains selectedfrom Protein A-based affibody libraries.” Prot, Struct. Funct. Gen.48:454-462; Gunneriusson et al., 1999, “Affinity maturation of a Taq DNApolymerase specific affibody by helix shuffling.” Prot. Eng. 12:873-878;Hansson et al., 1999, “An in vitro selected binding protein (affibody)shows conformation-dependent recognition of the respiratory syncytialvirus (RSV) G protein.” Immunotechnol. 4: 237-252; Henning et al., 2002,“Genetic modification of adenovirus 5 tropism by a novel class ofligands based on a three-helix bundle scaffold derived fromstaphylococcal protein A.” Human Gene Therapy 13:1427-1439; Högbom etal., 2003, “Structural basis for recognition by an in vitro evolvedaffibody. Proc Natl Acad Sci USA. 100(6):3191-3196; Nord et al., 1997,“Binding proteins selected from combinatorial libraries of an-helicalbacterial receptor domain.” Nature Biotechnol. 15:772-777; Nord et al.,2000, “Ligands selected from combinatorial libraries of protein A foruse in affinity capture of apolipoprotein A-1M and Taq DNA polymerase.”J. Biotechnol. 80:45-54; Nord et al., 1995, “A combinatorial library ofan alpha-helical bacterial receptor domain.” Prot. Eng. 8:601-608; Nordet al., 2001, “Recombinant human factor VIII-specific affinity ligandsselected from phage-displayed combinatorial libraries of protein A.”Eur. J. Biochem. 268:1-10; Nygren et al., 1997, “Scaffolds forengineering novel binding sites in proteins.” Curr. Opin. Struct. Biol.7:463-469; Rönnmark et al., 2002, “Human immunoglobin A (IgA)-specificligands from combinatorial engineering of protein A.” Eur. J. Bioehem.269:2647-2655; Rönnmark et al., 2002, “Construction and characterizationof affibody-Fc chimeras produced in Escherichia coli.” J, Immunol. Meth.261:199-211; Wahlberg et al., 2003, “An affibody in complex with atarget protein: structure and coupled folding.” Proc Natl Acad Sci USA.100(6):3185-3190; Gotz et al., 2002, “Ultrafast electron transfer in thecomplex between fluorescein and a cognate engineered lipocalin protein,a so-called anticalin.” Biochemistry. 41:4156-4164; Skerra, 2001,“Anticalins: a new class of engineered ligand-binding proteins withantibody-like properties.” J. Biotechnol. 2001 74:257-275; Skerra, 2000,“Lipocalins as a scaffold.” Biochim Biophys Acta. 1482:337-350; Skerraet al., 2000, “Engineered protein scaffolds for molecular recognition.”J Mol. Recognit. 13:167-187; Schlehuber et al., 2000, “A novel type ofreceptor protein, based on the lipocalin scaffold, with specificity fordigoxigenin.” J Mol. Biol. 297:1105-1120; Beste et al., 1999, “Smallantibody-like proteins with prescribed ligand specificities derived fromthe lipocalin fold.” Proc Natl Acad Sci USA. 96:1898-1903; PCTInternational Publication No. WO97/45538 entitled “Novel SyntheticProtein Structural Templates For The Generation, Screening And EvolutionOf Functional Molecular Surfaces” (relating to production of librariesof peptide sequences in the framework of a structural template derivedfrom Pleckstrin-Homology (PH) domains)).

Cancers that may be treated according to the invention include, withoutlimitation, any type of HNSCC or bladder cancer provided that theaffected cells exhibit increased expression of a protein that can betargeted at the cell surface. Tumors or tumor cells may be evaluated todetermine their susceptibility to the treatment methods of the inventionby, for example, obtaining a sample of tumor tissue or cells anddetermining the ability of the sample to bind to the ligand portion ofthe immunotoxin. In one embodiment, the protein on the cancer cells isEp-CAM. Cell-surface expression of Ep-CAM may be induced, or elevated,by an agent that increases steady-state levels of cell-surface Ep-CAM inpre-cancerous or cancerous tissue.

Accordingly, the present invention includes diagnostic methods and kitsthat can be used prior to the therapeutic method of the invention inorder to determine whether or not the HNSCC or bladder cancer expresseslevels of the protein that is bound by the ligand in the immunotoxin.Therefore, in a further embodiment, the present invention includes amethod for treating or preventing head and neck squamous cell carcinomaor bladder cancer comprising:

-   -   (1) testing a tumor sample from a patient for the expression of        a protein suspected of being associated with the head and neck        squamous cell carcinoma or bladder cancer; and    -   (2) if the protein is expressed at greater levels in the tumor        sample as compared to a control, administering to the patient an        effective amount of immunotoxin comprising:        -   (a) a ligand that binds to the protein on the cancer cell            attached to;        -   (b) a toxin that is cytotoxic to the cancer cell.

The present invention further includes a kit for diagnosing head andneck squamous cell carcinoma or bladder cancer comprising a ligand thatbinds to a protein on the cancer cell and instructions for the usethereof to diagnose the cancer.

In preferred non-limiting embodiments, the cancer is amenable totreatment by direct administration of the immunotoxin. For example, atarget tumor mass may be close to the surface of the skin. In anotherexample, a diseased tissue may be encapsulated by a cyst, or is found ina substantially enclosed cavity including, without limitation, a lumen(e.g., bladder). (Further details on direct administration are providedlater in the disclosure.)

In other embodiments, the cancer is amenable to treatment by intravenousadministration of the immunotoxin.

The invention also provides methods for reducing the risk ofpost-surgical complications comprising administering an effective amountof an immunotoxin before, during, or after surgery, and in specificnon-limiting embodiments, surgery to treat cancer.

The invention also provides methods for preventing occurrence,preventing or delaying recurrence, or reducing the rate of recurrence ofHNSCC or bladder cancer comprising directly administering to a patientin need thereof an effective amount of an immunotoxin.

The invention also provides methods for sensitizing a tumor or cancer toone or more other cancer therapeutics comprising administering animmunotoxin of the invention. In a nonlimiting embodiment, the othercancer therapeutic comprises another Ep-CAM-targeted immunotoxin. Inanother nonlimiting embodiment, the other cancer therapeutic comprisesradiation. The other cancer therapeutic may be administered prior to,overlapping with, concurrently, and/or after administration of theimmunotoxin. When administered concurrently, the immunotoxin and othercancer therapeutic may be administered in a single formulation or inseparate formulations, and if separately, then optionally, by differentmodes of administration. Accordingly, the combination of one or moreimmunotoxins and one or more other cancer therapeutics maysynergistically act to combat the tumor or cancer.

Where an immunotoxin of the invention is administered in addition to oneor more other therapeutic agents, these other cancer therapeutics mayinclude, without limitation, 2,2′,2″trichlorotriethylamme, 6-azauridine,6-diazo-5-oxo-L-norleucine, 6-mercaptopurine, aceglarone,aclacinomycinsa actinomycin, altretamine, aminoglutethimide,aminoglutethimide, amsacrine, anastrozole, ancitabine, angiogeninantisense oligonucleotide, anthramycin, azacitidine, azaserine,aziridine, batimastar, bcl-2 antisense oligonucleotide, benzodepa,bicalutamide, bisantrene, bleomycin, buserelin, busulfan, cactinomycin,calusterone, carboplatin, carboquone, carmofur, carmustine, carubicin,carzinophilin, chlorambucil, chloraphazine, chlormadinone acetate,chlorozotocin, chromomycins, cisplatin, cladribine, cyclophosphamide,cytarabine, dacarbazine, dactinomycin, daunorubicin, defosfamide,demecolcine, denopterin, diaziquone, docetaxel, doxifluridine,doxorubicin, droloxifene, dromostanolone, edatrexate, eflomithine,elliptinium acetate, emitefur, enocitabune, epirubicin, epitiostanol,estramustine, etoglucid, etoposide, fadrozole, fenretinide, floxuridine,fludarabine, fluorouracil, flutamide, folinic acid, formestane,fosfestrol, fotemustine, gallium nitrate, gemcitabine, goserelin,hexestrol, hydroxyurea, idarubicin, ifosfamide, improsulfan,interferon-alpha, interferon-beta, interferon-gamma, interleukin-2,L-asparaginase, lentinan, letrozole, leuprolide, lomustine, lonidamine,mannomustine, mechlorethamine, mechlorethamine oxide hydrochloride,medroxyprogesterone, megestrol acetate, melengestrol, melphalan,menogaril, mepitiostane, methotrexate, meturedepa, miboplatin,miltefosine, mitobronitol, mitoguazone, mitolactol, mitomycins,mitotane, mitoxantrone, mopidamol, mycophenolic acid, nilutamide,nimustine, nitracine, nogalamycin, novembichin, ollvomycins,oxaliplatin, paclitaxel, pentostain, peplomycin, perfosfamide, phenamet,phenesterine, pipobroman, piposulfan, pirarubicin, piritrexim,plicamycln, podophyllinic acid 2-ethyl-hydrazide, polyestradiolphosphate, porfimer sodium, porfiromycin, prednimustine, procabazine,propagermanium, PSK, pteropterin, puromycin, ranimustine, razoxane,roquinimex, sizofican, sobuzoxane, spirogermanium, streptonigrin,streptozocin, tamoxifen, tegafur, temozolomide, teniposlde, tenuzonicacid, testolacone, thiamiprine, thioguanine, Tomudex, topotecan,toremifene, triaziquone, triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide, trilostane, trimetrexate, triptorelin,trofosfamide, trontecan, tubercidin, ubenimex, uracil mustard, uredepa,urethan, vinblastine, vincristine, zinostatin, and zorubicin, cytosinearabinoside, gemtuzumab, thioepa, cyclothosphamide, antimetabolites(e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,5-fluorouracil, fludarabine, gemcitabine, dacarbazine, temozoamide),hexamethylmelamine, LYSODREN, nucleoside analogues, plant alkaloids(e.g., Taxol, paclitaxel, camptothecin, topotecan, irinotecan(CAMPTOSAR, CPT-11), vincristine, vinca alkyloids such as vinblastine.)podophyllotoxin, epipodophyllotoxin, VP-16 (etoposide), cytochalasin B,gramicidin D, ethidium bromide, emetine, anthracyclines (e.g.,daunorubicin), doxorubicin liposomal, dihydroxyanthracindione,mithramycin, actinomycin D, aldesleukin, allutamine, biaomycin,capecitabine, carboplain, chlorabusin, cyclarabine, daclinomycin,floxuridhe, lauprolide acetate, levamisole, lomusline, mercaptopurino,mesna, mitolanc, pegaspergase, pentoslatin, picamycin, riuxlmab,campath-1, straplozocin, tretinoin, VEGF antisense oligonucleotide,vindesine, and vinorelbine. Compositions comprising one or more cancertherapeutics (e.g., FLAG, CHOP) are also contemplated by the presentinvention. FLAG comprises fludarabine, cytosine arabinoside (Ara-C) andG-CSF. CHOP comprises cyclophosphamide, vincristine, doxorubicin, andprednisone. For a full listing of cancer therapeutics known in the art,see, e.g., the latest editions of The Merck Index and the Physician'sDesk Reference. Likewise, the immunotoxin of the invention may be usedin conjunction with radiation therapy or other known cancer therapeuticmodalities.

Pharmaceutical compositions for combination therapy may also include,without limitation, antibiotics (e.g., dactinomycin, bleomycin,mithramycin, anthramycin), asparaginase, Bacillus and Guerin, diphtheriatoxin, procaine, tetracaine, lidocaine, propranolol, anti-mitoticagents, abrin, ricinA, Pseudomonas exotoxin, nerve growth factor,platelet derived growth factor, tissue plasminogen activator,antihistaminic agents, anti-nausea agents, etc.

Indeed, direct administration of an effective amount of an immunotoxinto a patient in need of such treatment may result in reduced doses ofanother cancer therapeutic having clinically significant efficacy. Suchefficacy of the reduced dose of the other cancer therapeutic may not beobserved absent administration with an immunotoxin. Accordingly, thepresent invention provides methods for treating a tumor or cancercomprising administering a reduced dose of one or more other cancertherapeutics.

Moreover, combination therapy comprising an immunotoxin to a patient inneed of such treatment may permit relatively short treatment times whencompared to the duration or number of cycles of standard treatmentregimens. Accordingly, the present invention provides methods fortreating a tumor or cancer comprising administering one or more othercancer therapeutics for relatively short duration and/or in fewertreatment cycles.

Thus, in accordance with the present invention, combination therapiescomprising an immunotoxin and another cancer therapeutic may reducetoxicity (i.e., side effects) of the overall cancer treatment. Forexample, reduced toxicity, when compared to a monotherapy or anothercombination therapy, may be observed when delivering a reduced dose ofimmunotoxin and/or other cancer therapeutic, and/or when reducing theduration of a cycle (i.e., the period of a single administration or theperiod of a series of such administrations), and/or when reducing thenumber of cycles.

In a preferred embodiment, the invention provides methods for treatingand/or ameliorating the clinical condition of patients suffering fromHNSCC. Accordingly, the invention provides methods for (i) decreasingthe HNSCC tumor size, growth rate, invasiveness, malignancy grade,and/or risk of recurrence, (ii) prolonging the disease-free intervalfollowing treatment, and/or (iii) improving breathing, swallowing,and/or speech function in a patient with HNSCC, comprising administeringto the patient an effective amount of an immunotoxin. Clinicalimprovement may be subjectively or objectively determined, for exampleby evaluating the ability of a subject to breathe with less difficulty,the ability of the subject to swallow liquids versus solids, the degreeof obstruction, the quality or volume of speech, and other indices knownto the clinical arts.

In another preferred embodiment, the invention provides methods fortreating and/or ameliorating the clinical condition of patientssuffering from superficial transitional cell carcinoma of the bladder.Accordingly, the invention provides methods for (i) decreasing thebladder carcinoma tumor size, growth rate, invasiveness, malignancygrade, and/or risk of recurrence, (ii) prolonging the disease-freeinterval following other treatment, and/or (iii) curing the disease in apatient with transitional cell carcinoma of the bladder, comprisingadministering to the patient an effective amount of an immunotoxin.Clinical improvement may be determined, for example by cytologicalevaluation, cytoscopy or biopsy in a manner known to the clinical arts.

As mentioned previously, an immunotoxin of the invention comprises: (a)a ligand that binds to a protein on the cancer cell attached to; (b) atoxin that is cytotoxic to the cancer cell. The ligand may be “attached”to the target by any means by which the ligand can be associated with,or linked to, the toxin. For example, the ligand may be attached to thetoxin by chemical or recombinant means. Chemical means for preparingfusions or conjugates are known in the art and can be used to preparethe immunotoxin. The method used to conjugate the ligand and toxin mustbe capable of joining the ligand with the toxin without interfering withthe ability of the ligand to bind to the target molecule on the cancercell.

In one embodiment, the ligand and toxin are both proteins and can beconjugated using techniques well known in the art. There are severalhundred crosslinkers available that can conjugate two proteins. (See forexample “Chemistry of Protein Conjugation and Crosslinking”. 1991, ShansWong, CRC Press, Ann Arbor). The crosslinker is generally chosen basedon the reactive functional groups available or inserted on the ligand ortoxin. In addition, if there are no reactive groups a photoactivatiblecrosslinker can be used. In certain instances, it may be desirable toinclude a spacer between the ligand and the toxin. Crosslinking agentsknown to the art include the homobifunctional agents: glutaraldehyde,dimethyladipimidate and Bis(diazobenzidine) and the heterobifunctionalagents: m Maleimidobenzoyl-N-Hydroxysuccinimide and Sulfo-mMaleimidobenzoyl-N-Hydroxysuccinimide.

A ligand protein-toxin protein fusion may also be prepared usingrecombinant DNA techniques. In such a case a DNA sequence encoding theligand is fused to a DNA sequence encoding the toxin, resulting in achimeric DNA molecule. The chimeric DNA sequence is transfected into ahost cell that expresses the ligand-toxin fusion protein. The fusionprotein can be recovered from the cell culture and purified usingtechniques known in the art.

Preferably, the ligand binds to Ep-CAM, In one embodiment, theimmunotoxin comprises (a) an antibody or antibody fragment that binds toEp-CAM on the cancer cell attached to; (b) a toxin that is cytotoxic tothe cancer cells. (This immunotoxin is sometimes referred to as“Ep-CAM-targeted immunotoxin” herein.) In a specific embodiment, theimmunotoxin comprises (a) a humanized antibody or antibody fragment thatbinds to the extracellular domain of human Ep-CAM and comprisescomplementarity determining region (CDR) sequences derived from a MOC-31antibody attached to; (b) a toxin that is cytotoxic to the cancer cells.CDR sequences from the 4D5MOC-B antibody are shown in SEQ ID NOS:4-9.

Suitable Ep-CAM-targeted immunotoxins according to the inventioninclude, without limitation, VB4-845 and variants thereof, otherimmunotoxins that comprise the MOC31 variable region or variantsthereof, as well as immunotoxins that comprise other single or doublechain immunoglobulins that selectively bind Ep-CAM, or variants thereof.

In one embodiment, the Ep-CAM-binding portion comprises a completeimmunoglobulin molecule. In another embodiment, the Ep-CAM-bindingportion is a dimer of Fab, Fab′, scFv, single-domain antibody fragments,or disulfide-stabilized Fv fragments. In another embodiment, theEp-CAM-binding portion comprises a variable heavy chain, variable lightchain, Fab, Fab′, scFv, single-domain antibody fragment, ordisulfide-stabilized Fv fragment. Portions of the Ep-CAM-bindingmolecule may be derived from one or more species, preferably comprisingportions derived from the human species, and most preferably arecompletely human or humanized. Regions designed to facilitatepurification or for conjugation to toxin may also be included in oradded to the Ep-CAM-binding portion.

In a specific, non-limiting embodiment, the immunotoxin comprisesVB4-845 as shown in SEQ ID NO:2, In other non-limiting embodiments, theimmunotoxin comprises a variant of VB4-845. A VB4-845 variant binds tothe same Ep-CAM epitope or to a substantially similar Ep-CAM epitopethat is bound by VB4-845, and the variant may competitively inhibitVB4-845 binding to Ep-CAM, under physiologic conditions, by at least10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, or 95%. A VB4-845 variant may comprise the samePseudomonas exotoxin A fragment as VB4-845, or may comprise a differentportion of the same exotoxin or a different toxin.

In another non-limiting embodiment, the immunotoxin comprises anEp-CAM-binding portion comprising the variable region of MOC31, or avariant thereof. In yet another embodiment, the immunotoxin comprises anEp-CAM-binding portion comprising 4D5MOCB, or a variant thereof. Bindingof any of these immunotoxins to Ep-CAM may be reduced by at least 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, or 95% by competition with the reference MOC31 or 4D5MOCBantibody under physiologic conditions. The affinity of VB4-845 isK_(D)=1.6×10⁻⁸, using indirect flow cytometry on live cells.Lineweaver-Burke analysis (data Notebook: 0935, page 50) was performedusing method of Benedict et al (1997). J. Immunol. Methods, 201:223-231.The affinity of MOC31B, as described in Willuda et al (Cancer Research59, 5758-5767, 1999) is K_(D)=3.9×10⁻⁹, measured using RIA and Biacoreas described in methods. Consequently, the present invention includesimmunotoxins having a dissociation constant (K_(D)) of less than2.0×10⁻⁸.

Alternatively, the immunotoxin comprises an Ep-CAM-binding portion otherthan those discussed in the preceding paragraphs, but which selectivelybinds to Ep-CAM. In a preferred embodiment, the binding affinity of saidEp-CAM-binding portion is at least four orders of magnitude, preferablyat least three orders of magnitude, more preferably less than two ordersof magnitude of the binding affinity of VB4-845, PANOREX®, or MT-201 asmeasured by standard laboratory techniques. In non-limiting embodiments,the Ep-CAM-binding portion may competitively block the binding of aknown anti-Ep-CAM antibody, such as, but not limited to, PANOREX® orMT201, to Ep-CAM, under physiologic conditions, by at least 0.1%, 1%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, or 95%.

The skilled artisan would appreciate that specificity determiningresidues can be identified. The term “specificity determining residue,”also known as “SDR,” refers to a residue that forms part of the paratopeof an antibody, particularly CDR residues, the individual substitutionof which by alanine, independently of any other mutations, diminishesthe affinity of the antibody for the epitope by at least 10 fold,preferably by at least 100 fold, more preferably by at least 1000 fold.This loss in affinity underscores that residue's importance in theability of the antibody to bind the epitope. See, e.g., Tamura et al.,2000, “Structural correlates of an anticarcinoma antibody:identification of specificity-determining residues (SDRs) anddevelopment of a minimally immunogenic antibody variant by retention ofSDRs only,” J. Immunol. 164(3):1432-1441.

The effect of single or multiple mutations on binding activity,particularly on binding affinity, may be evaluated contemporaneously toassess the importance of a particular series of amino acids on thebinding interaction (e.g., the contribution of the light or heavy chainCDR2 to binding). Effects of an amino acid mutation may also beevaluated sequentially to assess the contribution of a single amino acidwhen assessed individually. Such evaluations can be performed, forexample, by in vitro saturation scanning (see, e.g., U.S. Pat. No.6,180,341; Hilton et al., 1996, “Saturation mutagenesis of the WSXWSmotif of the erythropoietin receptor,” J Biol. Chem. 271:4699-4708) andsite-directed mutagenesis (see, e.g., Cunningham and Wells, 1989,“High-resolution epitope mapping of hGH-receptor interactions byalanine-scanning mutagenesis,” Science 244:1081-1085; Bass et al., 1991,“A systematic mutational analysis of hormone-binding determinants in thehuman growth hormone receptor,” Proc Natl Acad. Sci. USA 88:4498-4502).In the alanine-scanning mutagenesis technique, single alanine mutationsare introduced at multiple residues in the molecule, and the resultantmutant molecules are tested for biological activity to identify aminoacid residues that are critical to the activity of the molecule.

Sites of ligand-receptor or other biological interaction can also beidentified by physical analysis of structure as determined by, forexample, nuclear magnetic resonance, crystallography, electrondiffraction, or photoaffinity labeling, in conjunction with mutation ofputative contact site amino acids (see, e.g., de Vos et al, 1992, “Humangrowth hormone and extracellular domain of its receptor: crystalstructure of the complex,” Science 255:306-312; Smith et al., 1992,“Human interleukin 4. The solution structure of a four-helix bundleprotein,” J Mol. Biol. 224:899-904; Wlodaver et al., 1992, “Crystalstructure of human recombinant interleukin-4 at 2.25 A resolution,” FEBSLett. 309:59-64. Additionally, the importance of particular individualamino acids, or series of amino acids, may be evaluated by comparisonwith the amino acid sequence of related polypeptides or analogousbinding sites.

Furthermore, the skilled artisan would appreciate that increased aviditymay compensate for lower binding affinity. The avidity of an immunotoxinfor Ep-CAM is an measure of the strength of the Ep-CAM-binding portion'sbinding of Ep-CAM, which has multiple binding sites. The functionalbinding strength between Ep-CAM and the Ep-CAM-binding portionrepresents the sum strength of all the affinity bonds, and thus anindividual component may bind with relatively low affinity, but amultimer of such components may demonstrate potent biological effect. Infact, the multiple interactions between Ep-CAM-binding sites and Ep-CAMepitopes may demonstrate much greater than additive biological effect,i.e., the advantage of multivalence can be many orders of magnitude withrespect to the equilibrium constant.

In one non-limiting embodiment, the Ep-CAM-binding portion has astructure substantially similar to that of 4D5MOCB. The substantiallysimilar structure can be characterized by reference to epitope maps thatreflect the binding points of the immunotoxin's Ep-CAM-binding portionto an Ep-CAM molecule.

Likewise, a variety of toxins may be used to design an Ep-CAM-targetedimmunotoxin according to the invention. In preferred embodiments, thetoxin comprises a polypeptide having ribosome-inactivating activityincluding, without limitation, gelonin, bouganin, saporin, ricin Achain, bryodin, diphtheria toxin, restrictocin, and variants thereof.When the protein is a ribosome-inactivating protein, the immunotoxinmust be internalized upon binding to the cancer cell in order for thetoxin to be cytotoxic to the cells.

In a particular preferred embodiment, the toxin portion comprises atleast a toxic portion of Pseudomonas exotoxin A (“ETA”), or a variantthereof. In a specific embodiment, the cytotoxic portion comprises anETA variant that, when administered alone, is substantially unable tobind to cells. In a further, specific embodiment, the cytotoxic portioncomprises ETA²⁵²⁻⁶⁰⁸. The cytotoxic portion may comprises one or morePseudomonas exotoxins known in the art (see, e.g., Kreitman, 1995,“Targeting pseudomonas exotoxin to hematologic malignancies,” Seminarsin Cancer Biology 6: 297-306; Pastan, 2003, “Immunotoxins containingpseudomonas exotoxin A: a short history,” Cancer Immunol. Immunother.52: 338-341), or variants thereof.

Several variants of Pseudomonas exotoxin, as well as methods of makingand using constructs comprising Pseudomonas exotoxin variants, are knownin the art (see, e.g., U.S. Patent Application No. US2003054012; U.S.Pat. No. 6,531,133; U.S. Pat. No. 6,426,075; U.S. Pat. No. 6,423,513;U.S. Pat. No. 6,074,644; U.S. Pat. No. 5,980,895; U.S. Pat. No.5,912,322; U.S. Pat. No. 5,854,044; U.S. Pat. No. 5,821,238; U.S. Pat.No. 5,705,163; U.S. Pat. No. 5,705,156; U.S. Pat. No. 5,621,078; U.S.Pat. No. 5,602,095; U.S. Pat. No. 5,512,658; U.S. Pat. No. 5,458,878;U.S. Pat. No. 5,082,927; U.S. Pat. No. 4,933,288; U.S. Pat. No.4,892,827; U.S. Pat. No. 4,677,070; U.S. Pat. No. 4,545,985;International Publication Nos. WO98/20135, WO93/25690; WO91/18100;WO91/18099; WO91/09949; and WO88/02401; Kondo et al, 19888, “Activity ofimmunotoxins constructed with modified pseudomonas exotoxin a lackingthe cell recognition domain.” J Biol. Chem. 263:9470-9475; Batra et al.,1989, “Antitumor activity in mice of an immunotoxin made withanti-transferring receptor and a recombinant form of pseudomonasexotoxin.” Proc Natl. Acad. Sci. USA 86:8545-8549; Puri et al., 1991,“Expression of high-affinity interleukin 4 receptors on murine sarcomacells and receptor-mediated cytotoxicity of tumor cells to chimericprotein between interleukin 4 and Pseudomonas exotoxin.” Cancer Res51:3011-3017; Siegall et al., 1992, “Cytotoxicity of chimeric (humanmurine) monoclonal antibody BR96 IgG, F(ab′)2, and Fab′ conjugated toPseudomonas exotoxin.” Bioconjug-Chem 3:302-307; Hall et al., 1994, “Invivo efficacy of intrathecal transferrin-Pseudomonas exotoxin Aimmunotoxin against LOX melanoma.” Neurosurgery 34:649-655; Kuan andPai, 1995, “Immunotoxins containing pseudomonas exotoxin that target Ley damage human endothelial cells in an antibody-specific mode: relevanceto vascular leak syndrome.” Clin Cancer Res 1:1589-1594; Kreitman, 1995,“Targeting pseudomonas exotoxin to hematologic malignancies.” Sem CancerBiol 6:297-306; Kawooya et al, “The expression, affinity purificationand characterization of recombinant pseudomonas exotoxin 40 (PE40)secreted from Escherichia coli.” J Biotechnol 42:9-22; Kaun and Pai,1995, “Immunotoxins containing pseudomonas exotoxin that target LeYdamage human endothelial cells in an antibody-specific mode: Relevanceto vascular leak syndrome.” Clin Cancer Res 1:1589-1594; Puri et al.,1996, “Preclinical development of a recombinant toxin containingcircularly permuted interleukin 4 and truncated Pseudomonas exotoxin fortherapy of malignant astrocytoma.” Cancer Res 56:5631-5637; Pai et al.,1996, “Treatment of advanced solid tumors with immunotoxin LMB-1: Anantibody linked to Pseudomonas exotoxin.” Nature Med. 3:350-353; Pai etal., 1998, “Clinical Trials with pseudomonas exotoxin immunotoxins.”Curr Top. Microbiol. Immunol. 234: 83-96; Klimka et al., 1999, “Ananti-CD30 single chain Fv selected by phage display and fused topseudomonas exotoxin A (Ki-4(scFv)-ETA′) is a potent immunotoxin againsta Hodgkin-derived cell line.” British J Cancer 80:1214-1222; Rand etal., 2000, “Intratumoral administration of recombinant circularlypermuted interleukin-4-Pseudomonas exotoxin in patients with high-gradeglioma.” Clin Cancer Res 6:2157-2165; Leland et al., 2000, “Human breastcarcinoma cells express type II IL-4 receptors and are sensitive toantitumor activity of chimeric IL-4-pseudomonas exotoxin fusion proteinin vitro and in vivo.” Molecular Medicine Today 6:165-178; Tur et al.,2001, “An anti-GD2 single chain Fv selected by phage display and fusedto Pseudomonas exotoxin A develops specific cytotoxic activity againstneuroblastoma derived cell lines.” Int J. Mol. Med 8:579-584; Onda etal., 2001, “Cytotoxicity of antiosteosarcoma recombinant immunotoxinscomposed of TP-3 Fv fragments and a truncated pseudomonas exotoxin A.” JImmunother 24:144-150; 18. “Synergistic interaction between ananti-p185her-2 pseudomonas exotoxin fusion protein [scfv(frp5)-eta] andionizing radiation for inhibiting growth of ovarian cancer cells thatoverexpress HER-2.” Schmidt et al., 2001, “Synergistic interactionbetween an anti-p185HER-2 pseudomonas exotoxin fusion protein[scFv(FRP5)-ETA| and ionizing radiation for inhibiting growth of ovariancancer cells that overexpress HER-2.” Gynecol Oncol 80:145-155; Pastan,2003, “Immunotoxins containing pseudomonas exotoxin A; a short history,”Cancer Immunol Immunother 52:338-341; Li et al, 1996, “Crystal structureof the catalytic domain of Pseudomonas exotoxin A complexed with anicotinamide adenine dinucleotide analog: implications for theactivation process and for ADP ribosylation.” Proc Natl Acad Sci USA.9:6902-6906; Kreitman and Pastan, 2003, “Immunobiological treatments ofhairy-cell leukaemia.” Best Pract Res Clin Haematol. 16:117-33.

In other nonlimiting embodiments, the toxin comprises an agent that actsto disrupt DNA. Thus, toxins may comprise, without limitation, enediynes(e.g., calicheamicin and esperamicin) and non-enediyne small moleculeagents (e.g., bleomycin, methidiumpropyl-EDTA-Fe(II)). Other toxinsuseful in accordance with the invention include, without limitation,daunorubicin, doxorubicin, distamycin A, cisplatin, mitomycin C,ecteinascidins, duocarmycin/CC-1065, and bleomycin/peplomycin.

In other nonlimiting embodiments, the toxin comprises an agent that actsto disrupt tubulin. Such toxins may comprise, without limitation,rhizoxin/maytansine, paclitaxel, vincristine and vinblastine,colchicine, auristatin dolastatin 10 MMAE, and peloruside A.

In other nonlimiting embodiments, the toxin portion of an immunotoxin ofthe invention may comprise an alkylating agent including, withoutlimitation, Asaley NSC 167780, AZQ NSC 182986, BCNU NSC 409962, BusulfanNSC 750, carboxyphthalatoplatinum NSC 271674, CBDCA NSC 241240, CCNU NSC79037, CHIP NSC 256927, chlorambucil NSC 3088, chlorozotocin NSC 178248,cis-platinum NSC 119875, clomesone NSC 338947,cyanomorpholinodoxorubicin NSC 357704, cyclodisone NSC 348948,dianhydrogalactitol NSC 132313, fluorodopan NSC 73754, hepsulfam NSC329680, hycanthone NSC 142982, melphalan NSC 8806, methyl CCNU NSC95441, mitomycin C NSC 26980, mitozolamide NSC 353451, nitrogen mustardNSC 762, PCNU NSC 95466, piperazine NSC 344007, piperazinedione NSC135758, pipobroman NSC 25154, porfiromycin NSC 56410, spirohydantoinmustard NSC 172112, teroxirone NSC 296934, tetraplatin NSC 363812,thio-tepa NSC 6396, triethylenemelamine NSC 9706, uracil nitrogenmustard NSC 34462, and Yoshi-864 NSC 102627.

In other nonlimiting embodiments, the toxin portion of an immunotoxin ofthe invention may comprise an antimitotic agent including, withoutlimitation, allocolchicine NSC 406042, Halichondrin B NSC 609395,colchicine NSC 757, colchicine derivative NSC 33410, dolastatin 10 NSC376128 (NG—auristatin derived), maytansine NSC 153858, rhizoxin NSC332598, taxol NSC 125973, taxol derivative NSC 608832, thiocolchicineNSC 361792, trityl cysteine NSC 83265, vinblastine sulfate NSC 49842,and vincristine sulfate NSC 67574.

In other nonlimiting embodiments, the toxin portion of an immunotoxin ofthe invention may comprise an topoisomerase I inhibitor including,without limitation, camptothecin NSC 94600, camptothecin, Na salt NSC100880, aminocamptothecin NSC 603071, camptothecin derivative NSC 95382,camptothecin derivative NSC 107124, camptothecin derivative NSC 643833,camptothecin derivative NSC 629971, camptothecin derivative NSC 295500,camptothecin derivative NSC 249910, camptothecin derivative NSC 606985,camptothecin derivative NSC 374028, camptothecin derivative NSC 176323,camptothecin derivative NSC 295501, camptothecin derivative NSC 606172,camptothecin derivative NSC 606173, camptothecin derivative NSC 610458,camptothecin derivative NSC 618939, camptothecin derivative NSC 610457,camptothecin derivative NSC 610459, camptothecin derivative NSC 606499,camptothecin 20 derivative NSC 610456, camptothecin derivative NSC364830, camptothecin derivative NSC 606497, and morpholinodoxorubicinNSC 354646.

In other nonlimiting embodiments, the toxin portion of an immunotoxin ofthe invention may comprise an topoisomerase II inhibitor including,without limitation, doxorubicin NSC 123127, amonafide NSC 308847, m-AMSANSC 249992, anthrapyrazole derivative NSC 355644, pyrazoloacridine NSC366140, bisantrene HCL NSC 337766, daunorubicin NSC 82151,deoxydoxorubicin NSC 267469, mitoxantrone NSC 301739, menogaril NSC269148, N,N-dibenzyl daunomycin NSC 268242, oxanthrazole NSC 349174,rubidazone NSC 164011, VM-26 NSC 122819, and VP-16 NSC 141540.

In other nonlimiting embodiments, the toxin portion of an immunotoxin ofthe invention may comprise an RNA or DNA antimetabolite including,without limitation, L-alanosine NSC 153353, 5-azacytidine NSC 102816,5-fluorouracil NSC 19893, acivicin NSC 163501, aminopterin derivativeNSC 132483, aminopterin derivative NSC 184692, aminopterin derivativeNSC 134033, an antifol NSC 633713, an antifol NSC 623017, Baker'ssoluble antifol NSC 139105, dichlorallyl lawsone NSC 126771, brequinarNSC 368390, ftorafur (pro-drag) NSC 148958, 5,6-dihydro-5-azacytidineNSC 264880, methotrexate NSC 740, methotrexate derivative NSC 174121,N-(phosphonoacetyl)-L-aspartate (PALA) NSC 224131, pyrazofurin NSC143095, trimetrexate NSC 352122, 3-HP NSC 95678,2′-deoxy-5-fluorouridine NSC 27640, 5-HP NSC 107392, alpha-TGDR NSC71851, aphidioclin glycinate NSC 303812, ara-C NSC 63878,5-aza-2′-deoxycytidine NSC 127716, beta-TGDR NSC 71261, cyclocytidineNSC 145668, guanazole NSC 1895, hydroxyurea NSC 32065, inosineglycodialdehyde NSC 118994, macbecin II NSC 330500, pyrazoloimidazoleNSC 51143, thioguanine NSC 752, and thiopurine NSC 755.

Furthermore, a cytotoxin may be altered to decrease or inhibit bindingoutside of the context of the immunotoxin, or to reduce specific typesof toxicity. For example, the cytotoxin may be altered to adjust theisoelectric point to approximately 7.0 such that liver toxicity isreduced.

Clinical outcomes of cancer treatments using an immunotoxin of theinvention are readily discernible by one of skill in the relevant art,such as a physician. For example, standard medical tests to measureclinical markers of cancer may be strong indicators of the treatment'sefficacy. Such tests may include, without limitation, physicalexamination, performance scales, disease markers, 12-Iead ECG, tumormeasurements, tissue biopsy, cytoscopy, cytology, longest diameter oftumor calculations, radiography, digital imaging of the tumor, vitalsigns, weight, recordation of adverse events, assessment of infectiousepisodes, assessment of concomitant medications, pain assessment, bloodor serum chemistry, urinalysis, CT scan, and pharmacokinetic analysis.Furthermore, synergistic effects of a combination therapy comprising theimmunotoxin and another cancer therapeutic may be determined bycomparative studies with patients undergoing monotherapy.

Particularly in the case of HNSCC, improvements in breathing,swallowing, speech, and certain quality of life measurements are readilyascertainable. Additionally, remission of HNSCC may be evaluated usingcriteria accepted by the skilled artisan. See, e.g., Therasse et al,2000, “New guidelines to evaluate the response to treatment in solidtumors. European Organization for Research and Treatment of Cancer,National Cancer Institute of the United States, National CancerInstitute of Canada,” J Natl Cancer Inst. February 2; 92(3):205-16.

The effective dose of immunotoxin to be administered during a cyclevaries according to the mode of administration. Direct administration(e.g., intratumoral injection) requires much smaller total body doses ofimmunotoxin as compared to systemic, intravenous administration of theimmunotoxin. It will be evident to the skilled artisan that localadministration can result in lower body doses, and in thosecircumstances, and resulting low circulating plasma level of immunotoxinwould be expected and desired.

Moreover, the effective dose of a specific immunotoxin construct maydepend on additional factors, including the type of cancer, the size ofthe tumour in the case of HNSCC, the stage of the cancer, theimmunotoxin's toxicity to the patient, the specificity of targeting tocancer cells, as well as the age, weight, and health of the patient.

In one embodiment, the effective dose by direct administration ofimmunotoxin may range from about 10 to 3000, 20 to 900, 30 to 800, 40 to700, 50 to 600, 60 to 500, 70 to 400, 80 to 300, 90 to 200, or 100 to150 micrograms/tumor/day. In other embodiments, the dose may range fromapproximately 10 to 20, 21 to 40, 41 to 80, 81 to 100, 101 to 130, 131to 150, 151 to 200, 201 to 280, 281 to 350, 351 to 500, 501 to 1000,1001 to 2000, or 2001 to 3000 micrograms/tumor/day. In specificembodiments, the dose may be at least approximately 20, 40, 80, 130,200, 280, 400, 500, 750, 1000, 2000, or 3000 micrograms/tumor/day.

In another embodiment, the effective dose of immunotoxin may range fromabout 100 to 5000, 200 to 4000, 300 to 3000, 400 to 2000, 500 to 1000,600 to 900, or 700 to 1500 micrograms/tumor/month. In other embodiments,the dose may range from approximately 100 to 199, 200 to 399, 400 to649, 650 to 999, 1000 to 1799, 1800 to 2499, 2500 to 3499, 3500 to 4999,5000 to 7499, 7500 to 10000, or 10001 to 20000 micrograms/tumor/month.In specific embodiments, the dose may be at least approximately 100,200, 400, 650, 1000, 1400, 2000, 2500, 3000, 3500, 4000, 4500, 5000,7500, 0000, or 20000 micrograms/tumor/month.

In another embodiment, the effective dose of immunotoxin results in anintratumoral concentration of at least approximately 5, 10, 20, 30, 40,50, 60, 75, 100, 125, 150, 100, 200, 300, 400, or 500 micrograms/cm³ ofthe immunotoxin. In other embodiments, the resulting intratumoralconcentration of immunotoxin is approximately 5 to 500, 10 to 400, 15 to300, 20 to 200, 25 to 100, 30 to 90, 35 to 80, 40 to 70, 45 to 60, or 50to 55 micrograms/cm³. In other embodiments, the resulting intratumoralconcentration of immunotoxin is approximately 10 to 15, 16 to 20, 21 to25, 26 to 30, 31 to 35, 36 to 40, 41 to 45, 46 to 50, 51 to 55, 56 to60, 61 to 65, 66 to 70, 71 to 75, 76 to 80, 81 to 85, 86 to 90, 91 to95, 96 to 100, or 100 to 200 micrograms/cm³.

In another embodiment, the effective dose of immunotoxin results in aplasma concentration of less than approximately 0.1, 1, 2.5, 5, 7.5, 10,15, 20, 30, 40, or 50 micrograms/liter. In other embodiments, theresulting circulating concentration of immunotoxin is approximately 0.1to 50, 1 to 40, 2.5 to 30, 5 to 20, or 7.5 to 10 micrograms/liter. Inother embodiments, the resulting circulating concentration ofimmunotoxin is approximately 0.1 to 1, 1.1 to 2.4, 2.5 to 5, 5.1 to 7.4,7.5 to 10, 11 to 15, 16 to 20, 21 to 30, 31 to 40, or 41 to 50micrograms/liter.

In a particular non-limiting embodiment, the effective dose of theimmunotoxin is between about 100 and 3000 micrograms/tumor/month, forexample approximately 100, 200, 300, 400, 750, or 1000micrograms/tumor/month, wherein the patient is administered a singledose per day. The single dose is administered approximately every monthfor approximately 1, 2, 3, 4, 5, or 6 consecutive months. After thiscycle, a subsequent cycle may begin approximately 1, 2, 4, 6, or 12months later. The treatment regime may include 1, 2, 3, 4, 5, or 6cycles, each cycle being spaced apart by approximately 1, 2, 4, 6, or 12months.

In a particular non-limiting embodiment, the effective dose of theimmunotoxin is between about 20 and 1240 micrograms/tumor/day, forexample approximately 20, 40, 80, 130, 200, or 280 micrograms/tumor/dayor approximately 100, 200, 330, 500, 700, 930, 1240micrograms/tumor/day, wherein the patient is administered a single doseper day. The single dose is administered approximately every day (one ormore days may optionally be skipped) for approximately 1, 2, 3, 4, 5, 6or 7 consecutive days. After this cycle, a subsequent cycle may beginapproximately 1, 2, 3, 4, 5, or 6 weeks later. The treatment regime mayinclude 1, 2, 3, 4, 5, or 6 cycles, each cycle being spaced apart byapproximately 1, 2, 3, 4, 5, or 6 weeks.

The injection volume preferably is at least an effective amount, whichis appropriate to the type and/or location of the tumor. The maximuminjection volume in a single dose may be between about 25% and 75% oftumor volume, for example approximately one-quarter, one-third, orthree-quarters of the estimated target tumor volume. In a specific,non-limiting embodiment, the maximum injection volume in a single doseis approximately 30% of the tumor volume.

In another embodiment, the immunotoxin is administered intratumourallyat a total dose per cycle equivalent to, or below the maximum tolerateddose established in a safety trial but the dosage is standardized inrelation to the tumour volume. For example, subjects will receivebetween 1 microgram per cm³ and 500 microgram per cm³ tumour or a dosesufficient to reach about between 14 picomole and 7 nanomole per cm³tumour tissue. The dose will be administered in a volume not exceedingabout 20-50% of the tumour volume. The immunotoxin will be diluted in asuitable salt solution. For example, for a tumour of estimated volume of3 cm³, a target dose of 14 picomoles (1 microgram per cm³), and amaximum injection relative volume of about ⅓ of the tumour, 3 microgramof immunotoxin will be diluted into about 1 ml of diluent.

In another particular embodiment, the effective dose of the immunotoxinis between about 20 and 300 micrograms/tumor/day, for exampleapproximately 20, 40, 80, 130, 200, or 280 micrograms/tumor/day, whereinthe patient is administered a single dose per day. The maximum injectionvolume in a single dose may be between about 25% and 75% of tumorvolume, for example approximately one-quarter, one-third, orthree-quarters of the estimated target tumor volume. The single dose isadministered every other day for approximately 5, 7, 9, 11, 13, 15, 17,19, 21, 23, 25, 27, 29, or 31 consecutive days. After this cycle, asubsequent cycle may begin approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, or 12 weeks later. The treatment regime may include 1, 2, 3, 4, 5,or 6 cycles, each cycle being spaced apart by approximately 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, or 12 weeks.

In one specific non-limiting embodiment, VB4-845 is administered at adose of approximately 280 micrograms/tumor/day, wherein the patient isadministered a single dose per day. The maximum injection volume in asingle dose is approximately one-third of the estimated target tumorvolume. The single dose is administered every day for approximately fiveconsecutive days. After this cycle, a subsequent cycle may beginapproximately one month later, preferably one month from the first dayof the first cycle. The treatment regime may include three cycles, eachcycle being spaced apart by approximately one treatment-free week.

In another specific non-limiting embodiment, VB4-845 is administered ata dose of approximately 280 micrograms/tumor/day, wherein the patient isadministered a single dose per day. The maximum injection volume in asingle dose is approximately one-third of the estimated target tumorvolume. The single dose is administered every other day forapproximately one week. After this cycle, a subsequent cycle may beginapproximately one week later. The treatment regime may include threecycles, each cycle being spaced apart by approximately one week.

In yet another specific embodiment, VB4-845 is administered at a dose ofapproximately 280 micrograms/tumor/day, wherein the patient isadministered a single dose per day. The maximum injection volume in asingle dose is approximately one-third of the estimated target tumorvolume. The single dose is administered every other day forapproximately three weeks. After this cycle, a subsequent cycle maybegin approximately one week later. The treatment regime may includethree cycles, each cycle being spaced apart by approximately one week.

For administration to a cavity such as the urinary bladder, theeffective dose of the immunotoxin is between about 100 and 2000micrograms in 50 ml/week (equivalent to a concentration of between about29 nanomolar to 580 nanomolar), for example approximately 100, 200, 335,500, 700, 930, 1240 micrograms in 50 ml/week, wherein the patient isadministered a single dose per week and the tumour tissue is exposed tothe immunotoxin for at least about 30 minutes. For example, the solutionis retained into the cavity for about 30 minutes to about 3 hours. In aspecific non-limiting embodiment, the tumour tissue is exposed to theimmunotoxin for about 1 hours or more preferably for about 2 hours.After this cycle, a subsequent cycle may begin approximately 1, 2, 4, 6,or 12 weeks after the previous dose. The treatment regime may include 1,2, 3, 4, 5, or 6 cycles, each cycle being spaced apart by approximately1, 2, 4, 6, or 12 months.

For smaller or larger cavities such as cysts or bladder substantiallysmaller or larger than average, the volume can be adjusted to ensureadequate exposure of the tissue without overextending the cavity. Wherethe volume needs to be adjusted, the effective dose of the immunotoxinshould be between about 20 and 600 nanomolar in concentration for atoxin with one binding site per molecule.

Dosage for the immunotoxin can also be expressed as molarity of thebinding site for the protein on the cancer cells. For example, theimmunotoxin VB4-845 has a molecular weight of about 69.7 kDa and onebinding site for Ep-Cam. It is known that other immunotoxin formats suchas divalent formats, Fab, Fab′ or (Fab′)₂ fragment could have adifferent molecular weight by virtue of the number of amino acids in thepolypeptide chain or chains. It is also known that for a similar formatone could alter the molecular weight by attaching additional groups tothe polypeptide such sugar moiety or polyethylene glycol. The use of adifferent toxin or a different variant of the toxin could also result inan immunotoxin with a different molecular weight than VB4-845 used inthe examples. Furthermore, changes to the polypeptide chain that resultin a longer or a shorter fragment could also be made and yet withoutlosing the binding of the immunotoxin to the chosen protein on thecancer cell. All those variations are contemplated in this application.As a result it may be helpful to express the dosage of the immunotoxinin terms of the number of moles of the binding sites for the protein onthe cancer cells. In the examples and the various embodiments, thedosages are expressed in micrograms and are based on the molecularweight of VB4-845. The following formula provides a simple way totransform micrograms into mole equivalent of binding sites; (1×10⁻⁶g/number of g per mole immunotoxin)×number of binding site perimmunotoxin molecule=Conversion Factor to go from microgram (1×10⁻⁶ g)of a given IT to moles of binding sites. For VB4-845, an immunotoxinwith only one binding site per molecule, the conversion would be done asfollows: Number of micrograms×14.3×10⁻¹² moles/microgram=number ofmoles.

For example, where 3000 micrograms are to be injected in a tumour on agiven day, 3000 micrograms×14.3×10⁻¹² moles/microgram=42.9×10⁻⁹ molesbinding sites (or 42.9 nanomoles or 42,900 picomoles). Where the dose isexpressed in terms of a concentration in a diluent or by tumour tissuevolume, one can transform the weight of the immunotoxin into moles andthen divide this number of moles by the volume of diluent where theresult can be expressed in terms of molarity or by the volume tumourtissue where the result can be expressed as moles per cm³ (or otherunits of volume) of tissue.

For example, where 1240 microgram are to be administered into thebladder in a volume of 50 ml: 1240 microgram×14.3×10⁻¹²moles/microgram=about 18×10⁻² moles binding sites and 18×10⁻¹² moles/50ml (or 0.05 liter)=about 355×10⁻⁹M (or 355 nanomolar).

The effective dose of another cancer therapeutic to be administeredtogether with an immunotoxin during a cycle also varies according to themode of administration. The one or more cancer therapeutics may bedelivered intratumorally, or by other modes of administration.Typically, chemotherapeutic agents are administered systemically.Standard dosage and treatment regimens are known in the art (see, e.g.,the latest editions of the Merck Index and the Physician's DeskReference).

For example, in one embodiment, the additional cancer therapeuticcomprises dacarbazine at a dose ranging from approximately 200 to 4000mg/m²/cycle. In a preferred embodiment, the dose ranges from 700 to 1000mg/m²/cycle.

In another embodiment, the additional cancer therapeutic comprisesfludarabine at a dose ranging from approximately 25 to 50 mg/m² cycle.

In another embodiment, the additional cancer therapeutic comprisescytosine arabinoside (Ara-C) at a dose ranging from approximately 200 to2000 mg/m²/cycle.

In another embodiment, the additional cancer therapeutic comprisesdocetaxel at a dose ranging from approximately 1.5 to 7.5 mg/kg/cycle.

In another embodiment, the additional cancer therapeutic comprisespaclitaxel at a dose ranging from approximately 5 to 15 mg/kg/cycle.

In yet another embodiment the additional cancer therapeutic comprisescisplatin at a dose ranging from approximately 5 to 20 mg/kg/cycle.

In yet another embodiment, the additional cancer therapeutic comprises5-fluorouracil at a dose ranging from approximately 5 to 20 mg/kg/cycle.

In yet another embodiment, the additional cancer therapeutic comprisesdoxorubicin at a dose ranging from approximately 2 to 8 mg/kg/cycle.

In yet another embodiment, the additional cancer therapeutic comprisesepipodophyllotoxin at a dose ranging from approximately 40 to 160mg/kg/cycle.

In yet another embodiment, the additional cancer therapeutic comprisescyclophosphamide at a dose ranging from approximately 50 to 200mg/kg/cycle.

In yet another embodiment, the additional cancer therapeutic comprisesirinotecan at a dose ranging from approximately 50 to 75, 75 to 100, 100to 125, or 125 to 150 mg/m2/cycle.

In yet another embodiment, the cancer therapeutic comprises vinblastineat a dose ranging from approximately 3.7 to 5.4, 5.5 to 7.4, 7.5 to 11,or 11 to 18.5 mg/m/cycle.

In yet another embodiment, the additional cancer therapeutic comprisesvincristine at a dose ranging from approximately 0.7 to 1.4, or 1.5 to 2mg/m²/cycle.

In yet another embodiment, the additional cancer therapeutic comprisesmethotrexate at a dose ranging from approximately 3.3 to 5, 5 to 10, 10to 100, or 100 to 1000 mg/m2/cycle.

Combination therapy with an immunotoxin may sensitize the cancer ortumor to administration of an additional cancer therapeutic.Accordingly, the present invention contemplates combination therapiesfor preventing, treating, and/or preventing recurrence of cancercomprising administering an effective amount of an immunotoxin prior to,subsequently, or concurrently with a reduced dose of a cancertherapeutic. For example, initial treatment with an immunotoxin mayincrease the sensitivity of a cancer or tumor to subsequent challengewith a dose of cancer therapeutic. This dose is near, or below, the lowrange of standard dosages when the cancer therapeutic is administeredalone, or in the absence of an immunotoxin. When concurrentlyadministered, the immunotoxin may be administered separately from thecancer therapeutic, and optionally, via a different mode ofadministration.

Accordingly, in one embodiment, the additional cancer therapeuticcomprises eisplatin, e.g., PLATINOL or PLATINOL-AQ (Bristol Myers), at adose ranging from approximately 5 to 10, 11 to 20, 21 to 40, or 41 to 75mg/m²/cycle.

In another embodiment, the additional cancer therapeutic comprisescarboplatin, e.g., PARAPLATIN (Bristol Myers), at a dose ranging fromapproximately 2 to 3, 4 to 8, 9 to 16, 17 to 35, or 36 to 75mg/m²/cycle.

In another embodiment, the additional cancer therapeutic comprisescyclophosphamide, e.g. CYTOXAN (Bristol Myers Squibb), at a dose rangingfrom approximately 0.25 to 0.5, 0.6 to 0.9, 1 to 2, 3 to 5, 6 to 10, 11to 20, or 21 to 40 mg/kg/cycle.

In another embodiment, the additional cancer therapeutic comprisescytarabine, e.g., CYTOSAR-U (Pharmacia & Upjohn), at a dose ranging fromapproximately 0.5 to 1, 2 to 4, 5 to 10, 11 to 25, 26 to 50, or 51 to100 mg/m²/cycle. In another embodiment, the additional cancertherapeutic comprises cytarabine liposome, e.g., DEPOCYT (Chiron Corp.),at a dose ranging from approximately 5 to 50 mg/m²/cycle.

In another embodiment, the additional cancer therapeutic comprisesdacarbazine, e.g., DTIC or DTICDOME (Bayer Corp.), at a dose rangingfrom approximately 15 to 250 mg/m²/cycle or ranging from approximately0.2 to 2 mg/kg/cycle.

In another embodiment, the additional cancer therapeutic comprisestopotecan, e.g., HYCAMTIN (SmithKline Beecham), at a dose ranging fromapproximately 0.1 to 0.2, 0.3 to 0.4, 0.5 to 0.8, or 0.9 to 1.5mg/m²/Cycle.

In another embodiment, the additional cancer therapeutic comprisesirinotecan, e.g., 25 CAMPTOSAR (Pharmacia & Upjohn), at a dose rangingfrom approximately 5 to 9, 10 to 25, or 26 to 50 mg/m2/cycle.

In another embodiment, the additional cancer therapeutic comprisesfludarabine, e.g., FLUDARA (Berlex Laboratories), at a dose ranging fromapproximately 2.5 to 5, 6 to 10, 11 to 15, or 16 to 25 mg/m²/cycle.

In another embodiment, the additional cancer therapeutic comprisescytosine arabinoside (Ara-C) at a dose ranging from approximately 200 to2000 mg/m²/cycle, 300 to 1000 mg/m²/cycle, 400 to 800 mg/m²/cycle, or500 to 700 mg/m²/cycle.

In another embodiment, the additional cancer therapeutic comprisesdocetaxel, e.g., TAXOTERE (Rhone Poulenc Rorer) at a dose ranging fromapproximately 6 to 10, 11 to 30, or 31 to 60 mg/m²/cycle.

In another embodiment, the additional cancer therapeutic comprisespaclitaxel, e.g., TAXOL (Bristol Myers Squibb), at a dose ranging fromapproximately 10 to 20, 21 to 40, 41 to 70, or 71 to 135 mg/kg/cycle.

In another embodiment, the additional cancer therapeutic comprises5-fluorouracil at a dose ranging from approximately 0.5 to 5mg/kg/cycle, 1 to 4 mg/kg/cycle, or 2-3 mg/kg/cycle.

In another embodiment, the additional cancer therapeutic comprisesdoxorubicin, e.g., ADRIAMYCIN (Pharmacia & Upjohn), DOXIL (Alza), RUBEX(Bristol Myers Squibb), at a dose ranging from approximately 2 to 4, 5to 8, 9 to 15, 16 to 30, or 31 to 60 mg/kg/cycle.

In another embodiment, the additional cancer therapeutic comprisesetoposide, e.g., VEPESID (Pharmacia & Upjohn), at a dose ranging fromapproximately 3.5 to 7, 8 to 15, 16 to 25, or 26 to 50 mg/m²/cycle.

In another embodiment, the additional cancer therapeutic comprisesvinblastine, e.g., VELBAN (Eli Lilly), at a dose ranging fromapproximately 0.3 to 0.5, 0.6 to 0.9, 1 to 2, or 3 to 3.6 mg/m²/cycle.

In another embodiment, the additional cancer therapeutic comprisesvincristine, e.g., ONCOVIN (Eli Lilly), at a dose ranging fromapproximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6 or 0.7 mg/m²/cycle.

In another embodiment, the additional cancer therapeutic comprisesmethotrexate at a dose ranging from approximately 0.2 to 0.9, 1 to 5, 6to 10, or 11 to 20 mg/m²/cycle.

In another embodiment, an immunotoxin is administered in combinationwith at least one other immunotherapeutic which includes, withoutlimitation, rituxan, rituximab, campath-1, gemtuzumab, and trastuzutmab.

In another embodiment, an immunotoxin is administered in combinationwith one or more anti-angiogenic agents which include, withoutlimitation, angiostatin, thalidomide, kringle 5, endostatin, Serpin(Serine Protease Inhibitor), anti-thrombin, 29 kDa N-terminal and a 40kDa C-terminal proteolytic fragments of fibronectin, 16 kDa proteolyticfragment of prolactin, 7.8 kDa proteolytic fragment of plateletfactor-4, a 13 amino acid peptide corresponding to a fragment ofplatelet factor-4 (Maione et al., 1990, Cancer Res, 51:2077-2083), a14-amino acid peptide corresponding to a fragment of collagen I (Tolmaet al., 1993, J. Cell Biol. 122:497-511), a 19 amino acid peptidecorresponding to a fragment of Thrombospondin I (Tolsma et al., 1993, J,Cell Biol. 122:497-511), a 20-amino acid peptide corresponding to afragment of SPARC (Sage et al., 1995, J. Cell Biochem. 57:1329-1334),and a variant thereof, including a pharmaceutically acceptable saltthereof.

In another embodiment, an immunotoxin is administered in combinationwith a regimen of radiation therapy. The therapy may also comprisesurgery and/or chemotherapy. For example, the immunotoxin may beadministered in combination with radiation therapy and cisplatin(Platinol), fluorouracil (5-FU, Adrucil), carboplatin (Paraplatin),and/or paclitaxel (Taxol). Treatment with the immunotoxin may allow useof lower doses of radiation and/or less frequent radiation treatments,which may for example, reduce the incidence of severe sore throat thatimpedes swallowing function potentially resulting in undesired weightloss or dehydration.

In another embodiment, an immunotoxin is administered in combinationwith one or more cytokines which include, without limitation, alymphokine, tumor necrosis factors, tumor necrosis factor-like cytokine,lymphotoxin, interferon, macrophage inflammatory protein, granulocytemonocyte colony stimulating factor, interleukin (including, withoutlimitation, interleukin-1, interleukin-2, interleukin-6, interleukin-12,interleukin-15, interleukin-18), and a variant thereof, including apharmaceutically acceptable salt thereof.

In yet another embodiment, an immunotoxin is administered in combinationwith a cancer vaccine including, without limitation, autologous cells ortissues, non-autologous cells or tissues, carcinoembryonic antigen,alpha-fetoprotein, human chorionic gonadotropin, BCG live vaccine,melanocyte lineage proteins, and mutated, tumor-specific antigens. Inyet another embodiment, an immunotoxin is administered in associationwith hormonal therapy. Hormonal therapeutics include, withoutlimitation, a hormonal agonist, hormonal antagonist (e.g., flutamide,tamoxifen, leuprolide acetate (LUPRON)), and steroid (e.g.,dexamethasone, retinoid, betamethasone, cortisol, cortisone, prednisone,dehydrotestosterone, glucocorticoid, mineralocorticoid, estrogen,testosterone, progestin).

In yet another embodiment, an immunotoxin is administered in associationwith a gene therapy program to treat or prevent cancer.

In yet another embodiment, an Ep-CAM-targeted immunotoxin isadministered in combination with one or more agents that increaseexpression of Ep-CAM in the tumor cells of interest. Ep-CAM expressionpreferably is increased so that a greater number of Ep-CAM molecules areexpressed on the tumor cell surface. For example, the agent may inhibitthe normal cycles of Ep-CAM antigen endocytosis. Such combinationtreatment may improve the clinical efficacy of the Ep-CAM-targetedimmunotoxin alone, or with other cancer therapeutics or radiationtherapy. In specific, nonlimiting embodiments, the agent which increasesEp-CAM expression in the tumor cells is vinorelbine tartrate (Navelbine)and/or paclitax (Taxol). See, e.g., Thurmond et al., 2003,“Adenocarcinoma cells exposed in vitro to Navelbine or Taxol increaseEp-CAM expression through a novel mechanism.” Cancer Immunol Immunother.July; 52(7):429-37.

Combination therapy may thus increase the sensitivity of the cancer ortumor to the administered immunotoxin and/or additional cancertherapeutic. In this manner, shorter treatment cycles may be possiblethereby reducing toxic events. Accordingly, the invention provides amethod for treating or preventing cancer comprising administering to apatient in need thereof an effective amount of an immunotoxin and atleast one other cancer therapeutic for a short treatment cycle. Thecycle duration may range from approximately 1 to 30, 2 to 27, 3 to 15, 4to 12, 5 to 9, or 6-8 days. The cycle duration may vary according to thespecific cancer therapeutic in use. The invention also contemplatescontinuous or discontinuous administration, or daily doses divided intoseveral partial administrations. An appropriate cycle duration for aspecific cancer therapeutic will be appreciated by the skilled artisan,and the invention contemplates the continued assessment of optimaltreatment schedules for each cancer therapeutic. Specific guidelines forthe skilled artisan are known in the art. See, e.g., Therasse et al.,2000, “New guidelines to evaluate the response to treatment in solidtumors. European Organization for Research and Treatment of Cancer,National Cancer Institute of the United States, National CancerInstitute of Canada,” J Natl Cancer Inst. February 2; 92(3):205-16.

Alternatively, longer treatment cycles may be desired. Accordingly, thecycle duration may range from approximately 10 to 56, 12 to 48, 14 to28, 16 to 24, or 18 to 20 days. The cycle duration may vary according tothe specific cancertherapeutic in use.

The present invention contemplates at least one cycle, preferably morethan one cycle during which a single cancer therapeutic or series oftherapeutics is administered. An appropriate total number of cycles, andthe interval between cycles, will be appreciated by the skilled artisan.The number of cycles may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, or 21 cycles. The interval between cyclesmay be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, or 21 days. The invention contemplates the continued assessmentof optimal treatment schedules for each immunotoxin and additionalcancer therapeutic.

In one nonlimiting embodiment of the invention, the immunotoxin isdirectly administered at high doses (e.g., a dose resulting in greaterthan approximately 100, 200, 300, 400, 500, or 1000 micrograms/cm³) forshorter periods. Accordingly, in one nonlimiting, specific embodiment,the immunotoxin is administered intratumorally at a dose that results inan intratumoral concentration of immunotoxin of at least approximately200, 300, 400, or 500 micrograms/cm³ once a week for two weeks.

An immunotoxin according to the invention may be comprised in apharmaceutical composition or medicament. Pharmaceutical compositionsadapted for direct administration include, without limitation,lyophilized powders or aqueous or non-aqueous sterile injectablesolutions or suspensions, which may further contain antioxidants,buffers, bacteriostats and solutes that render the compositionssubstantially isotonic with the blood of an intended recipient. Othercomponents that may be present in such compositions include water,alcohols, polyols, glycerin and vegetable oils, for example.Extemporaneous injection solutions and suspensions may be prepared fromsterile powders, granules and tablets. Immunotoxin may be supplied, forexample but not by way of limitation, as a lyophilized powder which isreconstituted with sterile water or saline prior to administration tothe patient.

Pharmaceutical compositions of the invention may comprise apharmaceutically acceptable carrier. Suitable pharmaceuticallyacceptable carriers include essentially chemically inert and nontoxiccompositions that do not interfere with the effectiveness of thebiological activity of the pharmaceutical composition. Examples ofsuitable pharmaceutical carriers include, but are not limited to, water,saline solutions, glycerol solutions, ethanol,N-(1(2,3-dioleyloxy)propyl)N,N,N-trimethylammonium chloride (DOTMA),diolesylphosphotidyl-ethanolamine (DOPE), and liposomes. Suchcompositions should contain a therapeutically effective amount of thecompound, together with a suitable amount of carrier so as to providethe form for direct administration to the patient.

In another embodiment, a pharmaceutical composition comprises animmunotoxin and one or more additional cancer therapeutics, optionallyin a pharmaceutically acceptable carrier.

The composition may be in the form of a pharmaceutically acceptable saltwhich includes, without limitation, those formed with free amino groupssuch as those derived from hydrochloric, phosphoric, acetic, oxalic,tartaric acids, etc., and those formed with free carboxyl groups such asthose derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc. In various embodiments of the invention, thepharmaceutical composition is directly administered to the area of thetumor(s) by, for example, local infusion during surgery, topicalapplication (e.g., in conjunction with a wound dressing after surgery),injection, means of a catheter, means of a suppository, or means of animplant. An implant can be of a porous, non-porous, or gelatinousmaterial, including membranes, such as sialastic membranes, or fibers.Suppositories generally contain active ingredients in the range of 0.5%to 10% by weight.

In other embodiments, a controlled release system can be placed inproximity of the target tumor. For example, a micropump may delivercontrolled doses directly into the area of the tumor, thereby finelyregulating the timing and concentration of the pharmaceuticalcomposition (see, e.g., Goodson, 1984, in Medical Applications ofControlled Release, vol. 2, pp. 115-138).

The present invention also provides a kit comprising an effective amountof an immunotoxin, optionally, in combination with one or more othercancer therapeutics, together with instructions for the use thereof totreat HNSCC or bladder cancer.

In accordance with one aspect of the present invention, the immunotoxinand/or other cancer therapeutic is delivered to the patient by directadministration. Accordingly, the immunotoxin and/or other cancertherapeutic may be administered, without limitation, by one or moredirect injections into the tumor, by continuous or discontinuousperfusion into the tumor, by introduction of a reservoir of theimmunotoxin, by introduction of a slow-release apparatus into the tumor,by introduction of a slow-release formulation into the tumor, and/or bydirect application onto the tumor. By the mode of administration “intothe tumor,” introduction of the immunotoxin and/or other cancertherapeutic to the area of the tumor, or into a blood vessel orlymphatic vessel that substantially directly flows into the area of thetumor, is also contemplated. In each case, the pharmaceuticalcomposition is administered in at least an amount sufficient to achievethe endpoint, and if necessary, comprises a pharmaceutically acceptablecarrier.

It is contemplated that the immunotoxin may be administeredintratumorally, whereas any other cancer therapeutic may be delivered tothe patient by other modes of administration (e.g., intravenously).Additionally, where multiple cancer therapeutics are intended to bedelivered to a patient, the immunotoxin and one or more of the othercancer therapeutics may be delivered intratumorally, whereas othercancer therapeutics may be delivered by other modes of administration(e.g., intravenously and orally).

In a particular, non-limiting embodiment, the immunotoxin and/or othercancer therapeutic may be administered by intratumoral injection, forexample, following the template shown in FIG. 1 (see Khuri et al, 2000,“A controlled trial of intratumoral ONYX-015, a selectively-replicatingadenovirus, in combination with cisplatin and 5-fluorouracil in patientswith recurrent head and neck cancer,” Nature Med. 6:879-885). Theimmunotoxin and/or other cancer therapeutic may be suspended comprisinga buffered aqueous solution, e.g., phosphate-buffered saline (“PBS”).The volume of the suspension comprising the immunotoxin may be less thanapproximately 5, 15, 25, 35, 45, 55, 65, 75, 85, or 95% of the estimatedvolume of the tumor mass to be injected. In specific embodiments, thevolume of the suspension comprising the immunotoxin is less thanapproximately 30, 40, or 50% of the estimated volume of the target tumormass,

With each administration of the immunotoxin and/or other cancertherapeutic, at least one puncture of the skin or oral mucosa is made ata site approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% of thedistance from the estimated tumor center to the tumor periphery.Administration of the immunotoxin by direct injection may result in oneor more needle tracks emanating radially from the center of the tumormass. In a particular, non-limiting embodiment, needle tracks may beoriented substantially as depicted in FIG. 1.

For bladder carcinoma, the immunotoxin can be introduced by catheter asdescribed in Example 15.

In a nonlimiting embodiment of the invention, medical imaging techniquesare used to guide the administration of the immunotoxin directly to thetumour. This is particularly useful in some tumour of the head and neckand in other types of tumour that are difficult to access. In thesecases, image guidance of the administration tool (needle, catheter, slowrelease apparatus, etc) are used to prevent damage to, or administrationinto critical anatomical structures such as blood vessels, nerve tract,etc. and to ensure that the immunotoxin is adequately distributedthroughout a three dimensional tumour. Medical imaging-guidancetechniques are well known to the medical art and comprise ultrasound, CTscans, X-ray and PET scan guidance.

The present invention will be better understood by the followingexemplary teachings. The examples set forth herein are not intended tolimit the invention.

EXAMPLES Example 1 VB4-845 Immunotoxin

VB4-845 is an immunotoxin comprised of a single-chain Fv recombinanthuman antibody fragment that is fused to a truncated form of Pseudomonasexotoxin A (ETA 252-608). The antibody fragment is derived from thehumanized MOC31 single-chain antibody fragment, 4D5MOCB, whichspecifically binds to Ep-CAM¹⁶⁻¹⁸.

Exotoxin A is one of the toxic proteins released by pathogenic strainsof Pseudomonas aeruginosa ¹⁹, It is secreted as a proenzyme with amolecular weight of 66,000 daltons²⁰. Exotoxin A is translocated intosusceptible mammalian cells, where covalent alteration of the moleculerenders it enzymatically active. Pseudomonas exotoxin A irreversiblyblocks protein synthesis in cells by adenositie diphosphate-ribosylatinga post-translationally modified histidine residue of elongationfactor-2, called diphthamide, and induces apoptosis⁴. The truncatedversion of ETA used in this construct, while still containing thedomains for inducing cell death, lacks the cell-binding domain, therebypreventing the ETA portion from entering cells absent targeting by theantibody portion of the immunotoxin.

The gene sequence encoding a truncated form of the ETA (ETA₂₅₂₋₆₀₈), andthe Ep-CAM-binding 4D5MOCB scFv sequence were used to construct VB4-845.The molecule contains both N- and C-terminal His₅ tails forpurification, as depicted in FIG. 2. The DNA and ammo acid sequence ofVB4-845 is depicted in FIGS. 3A-D and SEQ ID NOS:1 and 2. The Ep-CAMbinding portion is shown in SEQ ID NO:3. The CDR sequences are shown inSEQ ID NOS:4-9.

The resulting protein retains the specificity of the parent 4D5MOCB forEp-CAM. The expression vector for the protein, pING3302 (PlasmidpING3302 from Xoma Ireland Ltd was used for the construction of theexpression vector.) is carried and expressed by the E104 E. coli hoststrain. The protein is 648 amino acids in length and has a predictedmolecular weight of 69.7 kilodalton (kDa). In SDS-PAGE (sodium dodesylsulfate-polyacrylamide gel electrophoresis) analysis, VB4-845 isobserved as a single protein band of approximately 70 kDa. The proteinhas an isoelectric point (pI) of approximately 5.9, and is water-solubleforming a clear solution. Additional details regarding the preparationof VB4-845 are provided in Example 9, infra.

VB4-845 has been shown to specifically inhibit protein synthesis andreduce the viability of Ep-CAM-positive carcinoma cells in vitro. Asdemonstrated in Example 5, below, upon systemic administration to mice,VB4-845 inhibited growth and induced regression of tumor xenograftsderived from lung, colon, or squamous cell carcinomas. VB4-845 showedsimilar organ distribution as the parental single chain fragment (scFv)and preferentially localized to Ep-CAM-positive tumor xenografts with atumor:blood ratio of 5.4.

As demonstrated in Example 6, a peritumoral model in mice showedsignificant inhibition of tumor growth in VB4-845-treated animals. Infact, in this model, two mice with smaller tumor volumes (90 mm³) at thestart of treatment showed complete tumor regression and remained tumorfree during the experiment (see below). In all the efficacy studies, themice tolerated the treatments well, with no drug related mortality andno significant clinical observations suggestive of toxicity. These datasupport the direct administration of VB4-845 for targeted therapy ofsolid tumors.

The dose range per cycle of VB4-845 in humans may be 4micrograms/kilogram, i.e., 113 fold lower than the doses given to micein the efficacy studies, both in the intravenous and peritumoral models(see footnote 1 of Table 7): The monthly exposure in humans may beadministered as a micro-dose over the course of 5 days, with acumulative effect of 1 dose per week throughout the total tumor area.

Example 2 Dosage Forms and Compositions

VB4-845 has been studied as a nascent drug and has been found to beeffective in binding to tumor cell lines and in some model systems,preventing tumor growth. VB4-845 is formulated at 1 mg/ml in 20 mMsodium phosphate, 500 mM NaCl, pH 7.2, and can be administered by anintratumoral route with a 22-gauge needle. It is packaged in 1 mlborosilicate glass vials, closed with a gray butyl stopper and analuminum overseal. Two fill sizes are currently available; 0.1 and 0.2mL (0.1 mg and 0.2 mg VB4-845, respectively). Drug is stored at −70° C.The final product is not preserved and is for single use only.

The sample product is labeled, stored, and shipped according to writtenand approved standard operating procedures. The product may be shippedunder frozen conditions (e.g., on dry ice), and may be maintained, forexample, at the study site in a limited access, controlled −70° C.freezer that is monitored regularly for temperature.

The product may be maintained at this condition until time of use.

Example 3 Stability of VB4-845

The shelf-life of the product when stored at −70° C. is at least sixmonths. At physiological conditions (e.g., incubation of the drugproduct for four hours at 37° C. in PBS), the majority. of theimmunotoxin molecules (at least 91%) are still eluted as monomers of theappropriate molecular weight (approximately 70 kDa). The amount ofVB4-845 slowly decreases with time with no less than approximately 47%of the initial protein being present in monomeric form after twentyhours at 37° C. Similar results were obtained upon incubation of^(99m)Tc-labeled VB4-845 in human serum, further corroborating thesuitability of the immunotoxin for in vivo application.

Short term stability studies have been conducted to evaluate theinherent stability of the investigational product under routine handlingat the clinical site. VB4-845 was evaluated in its standard formulationat room temperature and at 2-8° C. In addition, VB4-845 was prepared ininjection buffer of phosphate-buffered saline with and without 800 mMurea and tested up to six hours at room temperature. The short termstability studies also evaluated the impact of repeated freeze-thawcycles on VB4-845.

VB4-845 was found to retain its biological activity over the course ofall the short-term stability studies. VB4-845 may be withdrawn from the−70° C. freezer the day of dosing and allowed to thaw at roomtemperature. VB4-845 may be prepared into the injection buffer in 4-6hours of its removal from the −70° C. storage condition. Once theproduct is formulated into the injection buffer of phosphate-bufferedsaline, the product may be injected into the patient within six hours ofpreparation. If the product cannot be used within a suitable timecourse, a new vial may be obtained from the inventory for dosing.

VB4-845 is stable in its original packaging for at least 20 hours atroom temperature, and if kept refrigerated (e.g., at 2-8° C.), for atleast 24 hours. If the product is unused, it can be refrozen for lateruse, particularly if the original container/closure system remainsintact.

Short term stability studies (up to 16 hrs incubation time) inbiological fluid including human plasma, serum and urine demonstratedthat VB4-845 retains it binding property and cell toxicity at least 16hrs.

Example 4 In Vitro Pharmacology

Studies have been conducted to determine the in vitro cytotoxicity ofVB4-845 to tumor cell cultures and in vivo efficacy models in animals.

To determine the ability of VB4-845 to specifically inhibit the growthof Ep-CAM-positive tumor cells, MTT(3-[4₅5-dimethylthiazol-2-yl]-2,5-disphenyltetrazolium bromide) assayswere performed⁵³. The MTT assay measures the viability of cells bymonitoring the reduction of the tetrazolium salt to formazan by enzymescontained only in live cells. Varying concentrations of VB4-845 wereadded to cell cultures and cell growth monitored over 72 hours.

VB4-845 is specifically cytotoxic against Ep-CAM-positive cell lines(e.g., HT29-colorectal carcinoma, MCF7-breast adenocarcinoma,CAL27-squamous cell carcinoma, SW2-small cell lung carcinoma) and doesnot affect the growth of the Ep-CAM-negative cell lines RL (e.g.,non-Hodgkin's lymphoma) and COLO320 (colorectal carcinoma). SW2, CAL27and MCF7 cells were found to be equally sensitive to the cytotoxiceffect of VB4-845 and their proliferation was inhibited with an IC₅₀ ofonly 0.005 pM. HT29 cells were found to be the least sensitive (IC₅₀ of0.2 _(P)M).

Pseudomonas exotoxin irreversibly inhibits protein synthesis inmammalian cells by ADP-ribosylation of elongation factor 2²¹⁻²². Todemonstrate that the cytotoxic activity of VB4-845 correlates with itsability to inhibit protein synthesis in Ep-CAM positive tumor celllines, the uptake of a radioactively labeled metabolite, [³H]leucine,into Ep-CAM positive SW2 cells was monitored⁵³.

Upon treatment of SW2 cells with VB4-845 for a total of thirty hours,protein synthesis was inhibited with an IC₅₀ of 0.01 pM. This effectshowed a similar dose response relationship to that previously measuredin the cytotoxicity assay. 25 Protein synthesis in the Ep-CAM-negativecontrol cell line, RL, was not affected.

Example 5 In Vivo Studies of Systemic Administration of VB4-845

Mice bearing large established Ep-CAM-positive SW2 (small cell lungcancer), HT29 (colorectal carcinoma) or CAL27 (HNSCC) tumor xenograftswere treated intravenously (i.v.) with VB4-845 using 1 of 2 differentdose regimens: 5 μg given every second day for 3 weeks (45 μg total); or10 μg given every second day for 1 week (30 μg total). Mice bearingEp-CAM-negative COLO320 tumor xenografts were used as controls. Tumorsize was monitored over the course of the study (33-51 days postinitiation of treatment)⁵³.

The results are summarized in Table 4. The mice tolerated the treatmentswell, with no drug related mortalities and no significant clinicalobservations suggestive of toxicity. As shown in FIG. 4, significantinhibition of the growth of all Ep-CAM-positive tumors was achieved bytreating mice with either dose schedule. Treatment of mice bearing SW2xenografts resulted in shrinkage of the tumor volume to maximal 20% ofthe initial size and a slight resumption of growth to a final 2.6-foldsize increase at the end of the monitored period. A similar effect wasachieved upon treatment of CAL27 tumors, which were reduced to maximal60% of the initial volume. Fifty days after start of the treatment, themedian tumor volume did not exceed 1.4-fold the initial size. Two ofseven mice treated with the 5 μg dose schedule showed complete tumorregression and remained tumor free. Neither CAL27 nor SW2 tumors showeda significant difference in their tumor response to the two treatmentschedules.

For HT29 tumors, strong growth inhibition (0.7-fold of the initialvolume) was achieved with the 5 μg dose schedule. As already observedfor CAL27 tumors, 3 of 7 mice showed complete regression of their HT29tumors. The efficacy of the 10 μg schedule was comparatively lower,indicating that for these tumors a long-term treatment is moreeffective. No antitumor effect of VB4-845 was seen in mice bearingEp-CAM-negative COLO320 control tumors.

Example 6 In Vivo Studies of Direct Administration of VB4-845

Athymic mice were injected subcutaneously (s.c.) into the lateral flankwith 10⁷ CAL27 HNSCC squamous cell carcinoma cells⁵⁴. After four weekswhen tumors had established, the mice were randomized into two groupswith an average tumor volume of 150 mm³ each. Eight mice were treated byperitumoral injection of VB4-845 at a dose of 5 μg given every secondday (Mon/Wed/Fri) for 3 weeks (total dose 45 μg). With each injectionthe 5 μg of immunotoxin were distributed into 2 to 3 injection spots.Control mice (n=5) remained untreated.

As summarized in Table 5, significant inhibition of tumor growth wasobserved in treated animals (FIG. 5). Two mice with smaller tumorvolumes (90 mm³) at the start of treatment showed complete tumorregression and remained tumor free during the experiment. No toxicitycould be observed during and after immunotoxin treatment.

Example 7 Biodistribution

In general, the literature indicates that scFv are cleared rapidly fromthe circulation, and give high tumor-to-background ratios (specificretention in tumor mass) at early time points in animal models²³⁻²⁵.T_(1/2) on average are 2-4 hours²⁶⁻²⁷, but can be longer (>8 hours)depending upon the construction of the molecule²⁸ and the route ofadministration. The highest uptake, depending on the molecule, tends tooccur in the kidneys and liver after systemic infusion.

The biodistribution of VB4-845 has been assessed in mice bearingestablished Ep-CAM-positive SW2 and Ep-CAM-negative COLO320 xenograftsat the contralateral flanks⁵³. The maximum dose of radiolabeled VB4-845detected in SW2 tumors was 2.93% ID/g after four hours, which thengradually decreased to 1.95% ID/g and 1.13% ID/g after at 24 and 48hours, respectively. In contrast, VB4-15 845 in COLO320 control tumorslocalized with a maximum dose of 1.65% ID/g after thirty minutes, whichthen rapidly declined to 1.06% ID/g after four hours and showed onlybackground levels after 48 hours.

VB4-845 showed a, slower blood clearance than the parental scFv. After24 hours, the total dose of VB4-845 in the blood was 0.42% ID/g, whichwas 1.5-fold more than the parent scFv (0.28% ID/g). Moreover,localization of the immunotoxin in SW2 tumors was also delayed comparedto the parent scFv, and the distribution of VB4-845 revealed atumor:blood ratio of 5.38 after 48 hours, which was comparable to theratio obtained with the scFv after 24 hours. At each time point, VB4-845preferentially accumulated in Ep-CAM-positive SW2 tumors compared toCOLO320 control tumor with a SW2:COLO320 ratio varying between 1.28 and2.95. This indicates that VB4-845 was retained in Ep-CAM-positive tumorsby specific antibody-antigen interactions and cellular uptake. Themarginal accumulation in COLO320 control tumors may be due to theincrease in vascular permeability often found in tumors. Analysis ofnormal tissues in these animals revealed that VB4-845 also localized inthe kidney, spleen, liver and to a lower extent in the bone.

Clinical observations made during the conduct of the pharmacokinetic andefficacy models in mice indicate that the product was well toleratedwithout any clinical signs indicative of toxicity. All animals livedthroughout the course of the studies and there was no drug relatedmortality.

Example 8 Toxicity Studies

A non-GLP study was performed to access the potential toxicity ofescalating doses of VB4-845 on the 3 tissues, liver, spleen and bone,seen to have the highest level of localization of radiolabeled VB4-845during the pharmacokinetic study.

Results are shown in Table 6.

VB4-845 was administered to immunocompetent C57BL/6 mice, which are moresensitive to wild type ETA-mediated liver damage than the athymic miceused in the previous efficacy models, VB4-845 was administered to themice i.v. at either 5 μg (250 μg/kg) or 10 μg (500 μg/kg) every otherday for three doses, or 20 μg (1000 μg/kg) every other day for twodoses. Twenty-four hours after the last dose, the activity of plasmatransaminase was determined and compared to mice treated with PBS (thus0 μg/kg VB4-845). No elevation of ALT/AST levels were observed in theplasma of mice 24 hours after completion of the 5 μg and 10 μg doseregimens (FIG. 6). Elevated transaminase activity was only observed uponadministration of the 20 μg dose. At the 24 hour post-dose timepoint,the animals were sacrificed and tissue specimens from the livers andspleens were stained by hematoxylin/eosin and analyzed by lightmicroscopy.

Consistent with the transaminase activity seen, only a few sites withnecrotic hepatocytes were found upon treatment with the 20 μg (1000μg/kg) immunotoxin dosing regimen, total exposure 40 μg (2000 μg/kg)(FIG. 7). No signs of histopathological changes or myelosuppression wereobserved at any dose in spleen and cellular components of whole bloodsamples.

A low starting dose of VB4-845 in humans may be 20 μg (0.29 μg/kg for a70 kg adult) given daily by micro-dose administration to differentsections of the tumor each day for five days, with a single cyclecumulative exposure of 100 μg/tumor (1.43 μg/kg for a 70 kg adult). Ahigher dose may be 280 μg (4.0 (μg/kg for a 70 kg adult) given in thesame fashion for a single cycle cumulative exposure of 1400 μg/tumor (20μg/kg for a 70 kg adult). On a body weight basis, the starting dose isapproximately 1585-fold less and the higher dose being 113-fold lessthan the monthly exposure by intravenous administration used in theabove-described mouse studies (Table 7). Based on this safety margin,such a dose range is considered to be safe with respect to the dosesused in a repeated fashion in mice that resulted in no clinicalobservations indicative of toxicity and the starting dose is 1056-foldlower than the monthly exposure to VB4-845 that showed no elevation intransaminase levels or histopathological changes in mice.

Single chain Fvs in rodents are cleared rapidly from the circulation,and give high tumor-to-background ratios (specific retention in tumormass) at early timepoints²³⁻²⁵, with t_(1/2) on average 2-4 hoursalthough this time can be longer²⁶⁻²⁷.

Similar to results obtained in animals, ¹²³I-labeled anti-CEA scFvdemonstrated a relatively short half-life, for example, 0.42(t_(1/2))and 5(t_(1/2)) hours in human patients²⁹. Tumor to blood ratiosincreased with time (5.6:1 at 24 hours, compared to 1-1.5:1 for wholeIgG anti-CEA antibody). Approximately 15-41% of the administeredradioactivity was excreted in the urine within the first 24 hours,suggesting that the kidneys are the primary organ of excretion. Activitywas seen in the liver after one hour, which activity decreased rapidlyover the next 21 hours, and was observed in the gall bladder, consistentwith biliary excretion of radionucleotide after liver catabolism ofantibody²⁹. A second study demonstrated a similar half-life of 0.32(t_(1/2)) and 10.59 (t_(1/2)) hours, respectively³⁰. The mean half-lifefor LMB-2, which is a scFv-ETA immunotoxin, varied from 173-494 minutes(monoexponential decay); however, this was partially related to diseaseburden in the peripheral blood and spleen³¹⁻³².

PK studies of VB4-845 administered to humans can be evaluated, and suchstudies may encompass not only unconjugated toxin levels, but also thoseof anti-VB4-845 antibodies (neutralizing antibodies), along withantibodies to the toxin (Pseudomonas exotoxin A) in plasma. The PK ofthe free circulating toxin may be assessed in every patient, preferablyhi the first cycle of treatment and follow-up. The neutralizingantibodies, and the anti-toxin antibodies may be assessed within thefirst cycle of treatment and follow-up. The time required to achievepeak circulating concentration (T_(max)) may he delayed due, forexample, to an intratumoral route of administration. Moreover, the peakcirculating concentration (C_(max)) may be reduced.

Monoclonal antibodies (“MABs”) directed against lumphoma-associatedantigens have been developed and clinically investigated for diagnosisand therapy of a number of human cancers. Toxicity relating to theadministration of MABs or antibody fragments to humans have beenreported, though primarily infusion related. Such toxicity events mayinclude fever, chills, nausea and headach¹⁴, uticaria, anaphylaxis,aseptic meningitis, hemolysis, leukopaenia, thrombocytopaenia, andvascular leak syndrome³³⁻³⁵. In come cases, these reactions may bepartly attributable to the patient's immune response to foreign protein,since most clinical trials have used murine, or murine/human chimericantibodies³³⁻³⁴.

In contrast, VB4-845 is a humanized protein. Furthermore, in a preferredroute of administration, intra- or peritumoral application of VB4-845may not result in as many toxicity events, or to be similar degree oftoxicity, as previously observed for other cancer immunotherapies.

Example 9 Preparation of VB4-845

Construction of the VB4-845 (also referred to as 4D5MOCB-ETA) expressionvector. The sequence encoding a truncated form of ETA (ETA252-608) wasamplified by PCR from plasmid pSW200⁶⁰ and cloned as an 1164 bpEcoRI-HindIII fragment downstream of the Ep-CAM-binding 4D5MOCB scFvsequence present in the pIG6-based⁶¹ 4D5MOCB scFv expression vector.⁶²The primers(Tox1:CTCGGAATTCGGTGGCGCGCCGGAGTTCCCGAAACCGTCCACCCCGCCGGGTTCTTCTGGTT TA (SEQID NO:10); Tox2: GTCAAGCTrCTACAGTTCGTCTTTATGGTGATGGTGGTGATGCGGCGGTTTCCCGGGCTG (SEQ ID NO: 11)) introduced an EcoRI restriction site betweenscFv and toxin and a C-terminal hexahistidine tag followed by theendoplasmic reticulum (ER) retention signal KDEL, a stop codon and aHindIII restriction site. To improve purity and yield during IMAC, asecond hexahistidine tag was added at the N-terminus between theperiplasmic signal sequence and the 4D5MOCB coding region. To this end,two pairs of oligonucleotides (Xbal 5′:CTAGATAACGAGGGCAAAAAATGAAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGGTTTCGCTACCGT (SEQ ID NO: 12); Xbal3′:GCCACTGCAATCGCGATAGCTGTCTTTTTCATTTT TTGCCCTCGTTAT (SEQ ID NO: 13);and EcoRV 5′: AGCOCAGOCCGACCACCATCATCACCATCACGAT (SEQ ID NO:14); EcoRV3′: ATCGTGATGGTGATGATGGTGGTCGGCCTGCGCTACGGTAGCGAAACCAGCCAGT (SEQ ID NO:15)) were heated to 8Q° C., allowed to anneal by gradually cooling toroom temperature and then ligated between the XbaI and EcoRV sites ofpIG6-4D5MOCBETAH6KDEL. The sequence was experimentally confirmed.

For periplasmic expression of VB4-845, the vector pIG6 was used, whichplaces the gene under lac promoter control in SB536, an E. coli straindevoid of the periplasmic proteases HhoA and HhoB.63. Five ml 2YT mediumcontaining ampicillin (100 mg/ml) were inoculated with a singlebacterial colony containing the VB4-845 (4D5MOCB-ETA) expression plasmidand grown overnight at 25° C. The bacteria were diluted in one liter of2YT medium supplemented with 0.5% glucose and ampicillin (100 mg/ml) toreach an A55O nm between 0.1 and 0.2 and transferred to 3-liter baffledshake flasks. The culture was further grown at 25° C. to an A550 nm of0.5 and immunotoxin production was induced for 4 h by adding a finalconcentration of 1 mM isopropyl-b-D-thiogalaetopyranoside (IPTG, Sigma).The harvested pellet derived from a bacterial culture with a final A550nm of 6 was stored at −80° C.

For purification, the pellet obtained from a one liter culture wasresuspended in 25 ml lysis buffer, containing 50 mM Tris-HCl (pH 7.5),300 mM NaCl, 2 mM MgSO₄ and supplemented with EDTA-free proteaseinhibitor cocktail (Roche Diagnostics, Mannheim, Germany) and DNase I.The bacterial suspension was lysed with two cycles in a French PressureCell press (SLS Instruments, Urbana, Ill.), centrifuged at 48,000 g in aSS-34 rotor for 30 min at 4° C. and subsequently filter-sterilized (0.22mm). The immunotoxin present in the cleared supernatant was purified bychromatography using a BIOCAD-System (Perseptive BioSystems) with aNi2+-iminodiacetic (IDA) column and a HQ/M-anion-exchange column coupledin-line as described in Pltückthun et al. 64 Before the lysate wasloaded, the Ni2+-IDA column was equilibrated with 20 mM Tris (pH 7.5),300 mM NaCl. After loading, the column was washed three times withdifferent salt solutions, all buffered with 20 mM Tris (pH 7.5), in theorder 300 mM, 510 mM and 90 mM NaCl. Subsequently, the column was washedwith 20 mM Tris (pH 7.5), 10 mM imidazole, 90 mM NaCl, before lie boundimmunotoxin was eluted with the same solution containing 200 mMimidazole (pH 7.5).

The eluate was directly loaded onto the HQ/M-anion-exchange column andthe bound immunotoxin was eluted with a salt gradient of 90-1000 mMNaCl, buffered with 20 mM Tris (pH 7.5). The fractions containing4D5MOCB-ETA were collected and concentrated using a 10 kDa cutoff filterby centrifugation at 2000 g and 4° C. (Ultrafree-MC low protein binding,Millipore). The quality of purified VB4-845 (4D5MOCB-ETA) was analyzedby a 10% SDS-polyacrylamide gel and Western blotting using a horseradishperoxidase (HRP)-conjugated anti-tetrahistidine antibody (QIAGEN,Hilden, Germany) diluted 1:5000 according to the manufacturer'srecommendations.

Analytical Gel Filtration and Determination of Thermal Stability.

Ten micrograms of purified VB4-845 (4D5MOCB-ETA) were diluted in 50 mlPBS pH 7.4 containing 0.005% Tween-20 and subsequently incubated at 37°C. Samples were analyzed at different time points (after 0 h, 2 h, 4 h,8 h, 10 h and 20 h) by gel filtration using the Smart system (Pharmacia,Uppsala) with a Superose-12 PC3.2/30 column. The column was calibratedin the same buffer with three protein standards: alcohol dehydrogenase(Mr 150,000), bovine serum albumin (Mr 66,000) and carbonic anhydrase(Mr 29,000). The same analytical setting was used to assess the thermalstability of the 99 mTc-labeled immunotoxin after a 20 h incubation at37° C. in human serum. The amount of immunotoxin monomers was determinedby g-scintillation counting of the eluted fractions.

Radiolabeling and Determination of Antigen-Binding Affinity.

VB4-845 (4D5MOCB-ETA) was radioactively labeled by stable site-specificcoordination of 99 mTc-tricarbonyl trihydrate to the hexahistidine tagspresent in the protein sequence.⁶⁵ This spontaneous reaction was inducedby mixing 30 ml of immunotoxin solution (1 mg/ml) with one third volumeof 1 M 2-[N-morpholino]ethanesulfonic acid (MES) pH 6.8 and one thirdvolume of freshly synthesized 99 mTc-tricarbonyl compound. The mixturewas incubated for 1 h at 37° C. and the reaction was stopped bydesalting over a Biospin-6 column (BioRad, Hercules, Calif.)equilibrated with PBS containing 0.005% Tween-20, according to themanufacturer's recommendation. The percentage of immunoreactiveimmunotoxin was assessed as described by Lindmo et al.⁶⁶ The bindingaffinity of the 99 mTc-labeled immunotoxin was determined on SW2 cellsin a radio-immunoassay (RIA), essentially as described for the scFv4D5MOCB.

Example 10 VB4-84S Manufacturing Process

VB4-845 E. coli Fermentation.

The production of VB4-845 is carried out in 2 L shake flasks using arotary incubator shaker in a research laboratory. The rotary shakerresides within an environmental control room where temperature can beregulated to within one degree Celsius. Inoculation of seed medium,production medium and all aseptic manipulations take place under abiological safety cabinet type II/B with HEPA filtration and airclassification of 100. Cell separation, concentration and diafiltrationtake place in a research laboratory.

VB4-845 is produced from the VB4-845 E104 host cell E. coli Master CellBank (MCB) (Plasmid pING3302 from Xoma Ireland Ltd was used for theconstruction of the expression vector.). Initial scale-up of cell(fermentation) propagation for the production of clinical grade VB4-845has been to the level of 26×2 L shake flasks with a working volume of 1L per flask, total volume is 26 L. The VB4-845 E. coli MCB is grown in acomplex nitrogen media containing glycerol as the principal carbonsources for cell growth. The fermentation procedure is described below.

Inoculum Preparation.

For a 26 L shake flask run, one 500 mL culture of VB4-845 E. coli MCB isprepared as pre-inoculum. For each culture, a vial of MCB is withdrawnfrom the −18° C. storage tank and allowed to thaw at room temperature.The vial is wiped externally with 70% ethanol and allowed to air dry ina biological safety cabinet. The cell suspension of MCB (1.5 ml) isadded to a 2 L Erlenmeyer flask containing 500 mL of sterile seed medium(modified 2YT medium and 25 mg/L tetracycline). The flask is transferredto a rotary shaker set at 200 rpm and grown at 25±1° C. until an opticaldensity of 3.0±0.2 or greater is reached (10.5±1 hr, mid-log phase ofgrowth). The inoculum is then used as a seed culture to inoculate the 26production shake flasks.

Fermentation in 26×2 L Shake Flasks.

Fermentation is carried out in 2 L-unbaffled flasks each containing 1 Lof production medium. A typical production run for clinical gradeVB4-845 has been 26×2 L flasks containing 1 L of production media(modified Terrific Broth, TB) per flask. The fermentation media isseeded with a 1% inoculum from the above culture and incubated on ashaker (200 rpm) at 25±1° C. until an optical density of 1.2 is reached(approximately 6-7 hours) at the last shake flask inoculated. A typicalOD600 range at induction is 1.2-1.5. The VB4-845 expression is inducedby the addition of 0.1% L-arabinose. Cells are harvested approximately 6hours post-induction.

Cell Separation.

At harvest, all shake flasks are removed from the shaker room in theorder of inoculation, with the first inoculated flask removed first. Thecontent of the first shake flask is added to the second shake flaskunder a biological hood. All subsequent shake flasks are removedlikewise. The pooled shake flasks are placed in refrigeration at 2-8° C.The VB4-845 E104 E. coli cells are removed in groups of 6 from the abovefermentation cultures by centrifugation at 6,800 g force for 15 minutesat 2-8° C. in a Sorvall and Beckman centrifuges. The cells are discardedwhile the cell free broth is retained for further processing. Theconcentrated cell suspension is collected, inactivated and disposed ofby established methods. The resulting supernatant is pooled and a 5 mlsample is reserved for product quantification. The centrifuges, rotorsand centrifuge bottles are thoroughly cleaned prior to processing thefermentation broth.

Concentration/Diafiltration.

Concentration and diafiltration of harvested culture supernatant isperformed by using a tangential flow Pellicon system with a Sartoriusmembrane (Hydrosart) molecular cut-off of 10 kD NMW (nominal molecularweight), and having a surface area of 3 square feet. The Pelliconfiltration system is thoroughly washed prior to usage. Concentration isperformed at a feed rate of 4 L/min and a permeate rate of 500 mL/min. A5 ml sample is taken at the final concentration step. Diafiltration isperformed against 0.02 M sodium phosphate, pH 7.2±0.2. Five volumechanges are required to achieve the desired conductivity of <10 mS. Thediafiltered concentrated product is clarified in a Sorvall centrifuge at6,800 g force for approximately 30 minutes at a set temperature of 2-8°C. The clear solution-containing product of interest is filtered priorto purification using a 0.22 μm dead-end filter. The clarification stepcomprises, after diafiltration, centrifugation, passage through 0.2 μmFilter, addition of Triton X-100, adjustment of conductivity, adjustmentof pH, and then follows purification.

VB4-845 Purification Procedures,

Purification of VB4-845 is performed in Viventia Biotech's Pilot Plant,a cGMP controlled area with HEPA filtration and controlled environmentalwith air Classification of 10,000. The VB4-845 protein is isolated bymetal-affinity chelating chromatography and is further purified by ananion exchange chromatography elution. The purification process issummarized in the flow diagram in FIG. 9, and is described below.

Chelating Sepharose Metal Interaction Chromatography.

The metal-affinity column is prepared by packing Chelating Sepharose HPresin in a XK26/20 glass column, with a column volume of approximately17±1 mL. The packing is performed at a backpressure of 3 bar. Theworking linear flow rate (LFR) is 90 cm/h. Five column volumes (CV) ofWater for Injection (WFI) are passed through the Chelating Sepharosecolumn. To charge the Chelating Sepharose column with metal ions, 5 CVof 0.1M nickel chloride solution is passed through the column. Theremainder of the unbound nickel chloride is washed away with 5 CV ofWFI. The column is then equilibrated with 10 CV of 20 mM sodiumphosphate containing 150 mM sodium chloride and 0.1% Triton X-100, pH7.2±0.1 buffer (chelating sepharose equilibration buffer).

The conductivity of the concentrated/diafiltered solution containingVB4-845 has been adjusted to 15±1 mS with sodium chloride and the pH isadjusted to 7.2±0.1 with 1M sodium hydroxide (NaOH). The VB4-845containing solution is applied to the Chelating Sepharose HP column at aLFR of 90 cm/Hr or 8 ml/min. The column then is washed with 20 CV ofwash buffer, 20 mM sodium phosphate, 150 mM sodium chloride, pH 7.2±0.1buffer containing 20 mM imidazole and 0.1% Triton X-100 (wash buffer).The VB4-845 is eluted from the column with six CV of 20 mM sodiumphosphate, 150 mM sodium chloride, pH 7.2±0.1 buffer, containing 500 mMimidazole (Chelating Sepharose elution buffer). The product is collectedin a 3 CV fraction starting from the beginning of the elution peak.

Q-Sepharose-Anion Exchange Chromatography.

The Q-Sepharose HP resin is packed in a XK16/20 glass column with afinal column volume of 5.0±0.5 mL. The operating linear flow rate is 156cm/h. The column is washed with 10 CV of WFI, then washed with 5 CV of1M sodium chloride in 20 mM sodium phosphate, pH 7.2±0.1 buffer andequilibrated with 10 CV 20 mM sodium phosphate, 90 mM sodium chloride,pH 7.2±0.1 buffer (2-sepharose equilibration buffer). The elution fromthe Chelating Sepharose column is diluted with 20 mM sodium phosphate,pH 7.2±0.1 buffer until a conductivity of 10±1 mS is achieved. Thepartially purified VB4-845 is loaded onto the Q-Sepharose column at aflow rate of 5.2 ml/min to further reduce endotoxin levels and DNA. Oncethe product has been bound, the anion exchange column is washed with 15CV of Q-Sepharose equilibration buffer. The contaminants are found inthe flow-through and wash steps. The product is eluted with 20 mM sodiumphosphate, 500 mM sodium chloride, pH 7.2±0.1 buffer as a 3 mL fraction.

Example 11 VB4-845 Competition Assay

The Ep-Cam-positive cell line CAL-27 (0.9×10⁶) is pre-incubated with anon-saturating amount of biotinylated-VB4-845 scFv (0.5-1.0 μg) for 10min at 4° C. in ice-cold PBS-5% FCS. After which, the test antibody(competitor) is diluted in ice-cold PBS-5% FCS and added to the mixtureat an amount equimolar to the amount non-biotinylated VB4-845 scFvcapable of completely inhibiting the binding of the biotinylated-VB4-845scFv. Following the incubation for 1 hr at 4° C., the cells are washedwith ice-cold PBS-5% FCS and incubated for an additional 30 min at 4° C.in the presence of Cy Chrome-conjugated streptavidin (Pharmingen, 1:120)diluted in wash buffer. The cells are washed at the end of theincubation period and analyzed by flow cytometry. As a negative control,CAL-27 tumor cells are incubated with 4B5 scFv, ananti-idiotype-specific scFv that reacts with the GD2-specific antibody14G2a but not with CAL-27, in place of VB4-845 scFv. Alternatively, anon-competitor (anti-HER-2/neu) that binds to CAL-27 is added in placeof 4B5 scFv. In either case, none to minimal change in medianfluorescence is detected from that measured for biotinylated-VB4-845scFv alone. For each antibody, the percent inhibition is calculatedaccording to the following equation:PI=[(F _(Max) −F _(Bgd))−(F _(T) −F _(Bgd))/(F _(Max) −F _(Bgd))]×100

wherein:

PI=percent inhibition; F_(max)=maximal median fluorescence withbiotinylated-VB4-845 scFv; F_(T)=median fluorescence ofbiotinylated-VB4-845 scFv in the presence of the test antibody;F_(Bgd)=background median fluorescence, the difference in medianfluorescence between biotinylated-VB4-845 scFv alone andbiotinylated-VB4-845 scFv in the presence of either of the negativecontrol antibodies. Also see Willuda et al., 1998. “High thermalstability is essential for tumor targeting of antibody fragments:engineering of a humanized anti-epithelial glycoprotein-2 (epithelialcell adhesion molecule) single-chain Fv fragment,” Cancer Res.59:5758-5767.

Example 12 HNSCC Clinical Trials

In two clinical trials in HNSCC aimed primarily at determining themaximum tolerated dose and at evaluating different dosing protocols,subjects with advanced HNSCC will receive intratumoral injection ofVB4-845 according to different dosing protocols. The starting dose (20micrograms/tumour/day for 5 days) represent less thanone-one-hundred-and-twentieth the highest single-dose intravenousexposure seen in 3-week mouse studies (on a body-surface-area basis) andless than one-seventieth the highest 5-day intravenous exposure in3-week mouse studies (on a body-surface-area basis).

The first trial (an open-label, single arm, safety and tolerabilitystudy) is ongoing and has completed or initiated the treatment of atleast 13 subjects. Two cycles of up to 130 microgram/tumour/day for 5days (dose level 4; 650 microgram per tumour per cycle, total exposureof 1300 microgram per tumour) have been completed in most of thesesubjects. In this trial, the drug is injected directly at the site ofthe tumour or into one of the secondary growths (metastases) in theregion of the head and the neck. The biggest or best accessible lesionis selected for injection (indicator or target lesion). The trialcomprises a 2-cycle dose escalation scheme. Each cycle is of 4-weeks or28-day duration. In the first 5 consecutive days of the cycle thesubjects receive daily intratumoral injections of the drug with astarting dose of 20 microgram/tumour/day for 5 consecutive days, thusproviding a 100 microgram per tumour per cycle (dose level 1). The 5-dayperiod is followed by a 23-days rest period during which no drug will beadministered. The subject will, however, undergo weekly follow-ups thatinclude clinical examinations and testing of blood and urine samples. Asecond 28-days cycle is then repeated before final evaluation. A minimumof 1 and up to 3 subjects are dosed in each dose level. The 6 doselevels are 100, 200, 400, 650, 1000, and 1400 microgram/tumour/cycle (or20, 40, 80, 130, 200 and 280 microgram/tumour/day for 5 consecutivedays). On the morning of dosing, each vial is be diluted with up to 9000μL of phosphate-buffered saline (PBS), and the required amount ofVB4-845 is drawn into the syringe. The dilution rate is adjusted toachieve a volume to be injected not exceeding about 30% of the estimatedvolume of the tumour mass to be injected. In the 5-day period and the 24hours following their last dose, the subjects are treated in anintensive care unit. Daily urine and blood 5 samples are taken tomonitor liver and kidney functions and determine drug concentration inblood. On each dosing event, a single puncture of the skin or oralmucosa is made at a site approximately 80% of the distance from thetumour center out to the tumour periphery, ensuring that the puncture isat a different site from previous punctures. Six (6) to 8 needle tracksemanating out radially from the puncture site are made and equal volumesof solution are injected into each area. Adequate analgesics will beadministered during treatment. Topical or systemic use ofcorticosteroids will be restricted to symptomatic skin or mucosaltoxicity of grade 3 or 4.

In a second trial the drug is also injected directly at the site of thetumour or into one of the secondary growths (metastases) in the regionof the head and the neck. If the subject has more than one lesion, themost accessible lesion approaching 5 cm in any greatest dimension willbe injected. If only small lesions are available, multiple lesions canbe treated for a combined greatest dimension of 5 cm. The subjects aredosed once a week, for four consecutive weeks. This 4-week period willbe followed by a 4-week rest period during which the condition of thesubject will be monitored. The initial dose level of VB4-845 is 100μg/tumour/week and the other dose levels are 200, 330, 500, 700, 930 and1240 microgram/tumour/week. On the morning of dosing, the vial(s)is(are) diluted with up to 800 μL of PBS, and then the required amountof VB4-845 is drawn into the syringe. The final volume to be injected isadjusted using a suspension volume of phosphate-buffered saline (PBS) soas not to exceed 30% of the estimated volume of the tumour mass to beinjected. The drug is to be administered so as to attempt to include theentire volume of the tumour on each dosing day. To administer, small (25to 27 gauge) needles attached to 1 cc Luer-lock syringes is insertedinto the base of the tumour at an approximately 45-degree angle.Depending on the size and location of the tumour, the injection may bedone by tracking the product through the tumour from a single puncturesite or by injecting the tumour from multiple sites, in 1 cm increments,in parallel rows approximately 0.5 to 1.0 cm apart and disbursingthroughout the tumour. Tumour response will be assessed before treatmentat the baseline visit and pre-dose at each subsequent dose, at week 4and at the end of the study. Where possible, a CT-scan will be performedat Screening, Week 4 and Week 8 (or final visit). In the event that acomplete or partial response is observed, a CT scan will be performed 4weeks later to confirm the result. Other assessments will be by directmeasurement of the tumour by clinical observation and manipulation. AComplete response (CR) would indicate the complete disappearance of theinjected tumour (confirmed at 4 weeks); Partial response (PR) areduction by at least 30% in the largest diameter of the treated tumour(confirmed at 4 weeks); Stable disease by a regression of the treatedtumour of less than 30% or progression less than 20% and Tumourprogression (TP) an increase by 20% in the largest diameter of thetreated tumour, where CR, PR or SD have not been previously documented.Pain will be assessed using an analog pain scale before treatment andprior to each dose and at Week 4 and Week 8 (or final visit). Randomfine needle aspirate biopsies of the target tumour will be taken toexplore the effects of VB4-845 at a cellular level. Systemic and localtoxicity will be assessed using standard procedures and ongoingevaluations for adverse events, laboratory toxicities and subject painstatus will occur throughout the treatment.

Example 13 Biological Activity of VB4-845 Against Bladder Tumor CellLines

Summary

VB4-845 [anti-Ep-CAM scFv and Pseudomonas exotoxin A lacking the cellbinding domain (ETA252-608) fusion protein] was assessed by flowcytometry for cell-surface reactivity against a panel of human tumorcell lines including 14 bladder cancer cell lines to determine thedegree and broadness of Ep-CAM expression in this potential clinicalindications. VB4-845 demonstrated strong reactivity against 10 of 14bladder cancer cell lines and weak reactivity against one other. VB4-845demonstrated strong cytotoxicity on eleven VB4-845-positive bladdercancer cell lines; the IC50 values varied from 0.001-320 μM for a 72hour exposure. In contrast, no cytotoxicity was detected against thethree VB4-845-negative cell lines. Four bladder cancer cell lines (T-24,SW-870 UM-UC-10 and 1A6) were determined to be the most sensitive toVB4-845 treatment. In another experiment on a subset of cell lines wherethe exposure time was limited to 2 hours, VB4-845 exerted effectivecytotoxicity (>93%) against the squamous bladder cancer cell line,SCaBER and the transitional bladder carcinoma cell line, 5637. Incontrast, for a 2 hour exposure of 5637 to the control immunotoxin,4B5-PE, non-specific cytotoxicity was shown to be minimal (<10%) at 500pM and remained at the same level even after increasing the dose100-fold (50000 pM). In summary, the potent in vitro antitumor activityof VB4-845 on bladder cancer cell lines suggests that VB4-845 hasutility for pre-clinical and clinical development of anti-cancer therapyagainst bladder cancers.

Experimental Design

The experimental design for testing the reactivity of VB4-845 to tumorcell lines by flow cytometry and cytotoxicity have been described 3,4.Purified scFv-ETA fusion proteins, VB4-845 (Lot #02203, 1 mg/mL) and thenegative control 4B5scFv-ETA (Lot #032403, 1.5 mg/mL) were generated asdescribed and stored in aliquots at −80° C. The panel of tumor celllines used in the study and their characteristics are in Table 9. Alltumor cells were propagated in culture medium containing 10-20% FCS andappropriate supplements, following ATCC or ECACC protocols. Tumor cellswere harvested when the cultures were 50-70% confluent with viabilitygreater than 90%. The cell line CAL-27 expresses a high level of Ep-CAMantigen and was used as the positive control, while the low Ep-CAMexpressing cell line COLO 320 was used as the negative control.

Testing Reactivity of VB4-845 Against Tumor Cell Lines by Flow Cytometry

Purified VB4-845 was tested against the panel of tumor cell lines todetermine the cell-surface reactivity by flow cytometry. Briefly, tumorcells (0.9×106/300 μL) were incubated with purified VB4-845 or 4B5 scFvas a negative control, at 10 μg/mL for 2 hours on ice. Anti-EGFR mousemonoclonal antibody (Oncogene Research, Cat # OP15, at 1 μg/mL) was usedas a positive control. After incubation, the cells were washed withPBS-5% FBS and incubated with either anti-HIS-Tag antibody (AmershamPharmacia Cat #27-4710-01, diluted 1:800) for VB4-845 orbiotin-conjugated anti-mouse IgG for anti-EGFR (Pierce cat #31174,diluted 1:200) for 1 hour on ice. The cells were washed with PBS-5% FBS,followed by incubation with either FITC-conjugated goat anti-mouse IgG(The binding Site Cat # AF271, diluted 1:100, for anti-HIS treatedcells), or Streptavidin-Cy-Chrome (Pharmingen cat#13038A, diluted 1:120)for 30 minutes on ice. Finally, the cells were washed and resuspended in0.5 mL of buffer containing propidium iodide (Molecular Probes cat#P-1304) at 0.6 μg/mL. Tumor cell binding was determined using aFACSCalibur. Antibodies were considered positive if antibody-treatedtumor cells exhibited a positive shift in fluorescence resulting in >30%positive cells (1.3 times control) over the negative control.

Assessment of VB4-845-Mediated Cytotoxicity by Cell Proliferation Assay

VB4-845 cytotoxicity was measured by determining inhibition of cellproliferation by an MTS assay. Briefly, 96-well microtitre plates wereprepared by seeding tumor cells at 5000 cells/50 μL/well in culturemedium containing 10% FCS. The plates were incubated for 3 hours at 37°C. in the presence of 5% CO2. Ten-fold serial dilutions of VB4-845 weremade at this time and varying amounts of VB4-845 (0.00005 to 500 μM)were added to each well in a 50 μL, volume, to bring the final volume to100 μL. As a negative control, 4B5 scFv-ETA was used at the sameconcentrations. The control cells and the control (empty) wells wereincubated with 100 μL of medium only, in quadruplets. The plates wereincubated for 72 hours at 37° C. in the presence of 5% CO2. Each assaywas repeated twice to demonstrate reproducibility and consistency inresults. After incubation, an MTS assay was performed to measure cellviability. Briefly, 75 μL of phenazine methosulfate, PMS (0.92 mg/mL inPBS) was added to 1.5 mL of a tetrazolium compound, MTS (Promega, Cat #G111A and G109C, 2 mg/mL in PBS) and 20 μL of the PMS/MTS mixture wasadded to each well. The plates were incubated for 2 hours at 37° C. inthe presence of 5% CO2. Subsequently, the plates were read at 490 nmusing an ELISA plate reader.

Determination of the Minimal Immunotoxin Exposure Time Required forVB4-845-Mediated Cytotoxicity

The IC50 (the VB4-845 concentration that kills fifty percent of cellscompared to cells treated with medium only) was determined by exposingVB4-845 to each bladder cancer cell line for 72 hours. Five-sensitivecell lines of varying sensitivity to killing (SW-780, UC-MC-10, 1A6,UC-MC-14 and 5637) were selected to establish the minimal exposure timerequired for killing of 50% tumor cells using a fixed concentration thatapproximated the IC50. Tumor cells were exposed to VB4-845 at a fixedconcentration (0.01, 0.6 or 6 μM) for 2, 4, 24, 48 and 72 hours. Exceptfor 72 hours, at each time point, the medium containing VB4-845 wasreplaced with a fresh culture medium to minimize the immunotoxin(VB4-845) exposure time. MTS assay was performed after 72 hours ofincubation to determine the cytotoxicity (50% tumor cell killing)compared to the control (cells with medium only). To further evaluatethe effect of VB4-845 on a less sensitive squamous cell carcinoma cellline (SCaBER) and a sensitive a bladder transitional carcinoma cell line(5637) and two other bladder cancer cell lines (UM-UC-10 and UM-UC-14),cells were exposed to 2 hours with either fixed concentration or varyingdoses of VB4-845. After 2 hours of incubation, cells were washed toremove VB4-845, incubated with fresh medium and MTS assay was performedafter 72 hours. Furthermore, to establish the specific cytotoxic effectof VB4-845, 5637 cells were exposed to varying doses (500, 5000, 50000pM) of VB4-845 and the negative control immunotoxin, 4B5-PE to determinethe effect of killing at higher concentrations. After incubation at eachtime points, 2, 6, 12, 24 and 48 hours, cells were washed with medium toremove VB4-845, incubated with fresh 1 medium and MTS assay wasperformed after 72 hours to determine cytotoxicity. The dose range wasselected on the basis of initial IC50 results with the expectations thatthe minimal dose of VB4-845 being used to give maximal killing of thiscell line.

Results

VB4-845 Tumor Cell Reactivity

The cell-surface reactivity of VB4-845 was assessed against a panel ofbladder tumor cell lines. VB4-845 demonstrated positive reactivityagainst 11 of the 14 bladder cancer cell lines cell lines. The data aresummarized in Table 10.

Cytotoxic Effect of VB4-845 Against Bladder Cancer Cell Lines In Vitro

Tumor cells were incubated with VB4-845 for 72 hours at concentrationsranging from 0.00005 to 500 pM and inhibition of cell proliferation wasassessed by MTS assay. Results are summarized in Table 10. VB4-845 didnot inhibition of cell proliferation in the three EGP-2-negative celllines (J-82, UM-UC-3 and UM-UC-13) but showed strong inhibition (IC50from 0.001-0.033 μM) in the four cell lines with very high expression ofEp-CAM antigen (T-24, SW-780, UM-UC-10 and 1A6 and intermediateinhibition in the other cell lines.

Minimal Exposure Time Required to Achieve VB4-845-Mediated CytotoxicityAgainst Bladder Cancer Cell Lines In Vitro

In the standard cell proliferation assay, bladder carcinoma cells wereexposed to VB4-845 for 72 hours, after which the inhibition of cellproliferation was assessed. For bladder cancer, intravesical therapydwell times are seldom longer than two hours. Therefore, a cellproliferation assay was performed to determine the minimal exposure timerequired to kill 50% tumor cells upon exposure to VB4-845 at a fixedconcentration at or near IC50 (0.01 or 0.6 pM). In the first experiment,two VB4-845-sensitive bladder cancer cell lines (SW-780 and 1A6) wereexposed to VB4-845 at 0.01 pM concentration for 2, 4, 6, 24, 48 or 72hours. VB4-845 demonstrated strong cytotoxicity on the SW-780 and 1A6bladder cancer cell lines even after short exposure time. For the highlysensitive bladder cancer cell line SW-780, (with an IC50 0.002 pM), 50%tumor cells were killed after 3 hours of exposure, whereas for a lesssensitive cell line, 1A6, (IC50 0.033 pM), the same was achieved after37 hours of exposure. A similar set of data was obtained in the secondexperiment, after exposure of three different bladder cancer cell linesto VB4-845 at 0.6 pM concentration. The results indicated that 50% ofUM-UC-10, 5637 or UM-UC-14 cells were killed after 4, 16 and 20 hours ofexposure, respectively. The rank order of sensitivity of these threelines was the same as for their IC50.

In a separate experiment, upon exposure to VB4-845 at a higherconcentration (6.0 pM) for 2 hours, 96, 89 and 93% of UM-UC-10, 5637 andUM-UC-14 cells were killed, respectively. On further evaluation, afterexposing a less-sensitive cell line (SCaBER) and a sensitive cell line(5637) for 2 hours with a varying dose of VB4-845, a strong cytotoxiceffect with >93% killing of SCaBER cells was achieved with a 3900 pMdose, when the same degree of cytotoxicity was achieved with a muchlesser dose (<498 pM) for 5637 cells. Thus, it was confirmed that theminimal dose of VB4-845 required for achieving maximal cytotoxic effectis dependent on the sensitivity of the cell line. Furthermore, in aseparate experiment, exposure of 5637 to VB4-845 for 2 hours at 500 pMconcentration demonstrated effective killing (>93%) of the cells with aminimal non-specific cytotoxicity (<10%) being demonstrated by thecontrol immunotoxin (4B5-PE). In fact, for a 2 hour exposure,nonspecific killing was kept to a minimal level even after increasingthe 4B5-PE concentration 100-fold.

Example 14 Human Clinical Bladder Binding

Surgical and necropsy human bladder tissue specimens were obtained andtested for Ep-CAM binding using VB4-845. The specimens wereformalin-fixed and paraffin-imbedded. Method validation was conducted onboth fresh-frozen and on fixed samples to confirm the adequacy of fixedspecimen for this assay and to determine the optimal antibody (VB4-845)concentration to use (minimizing non-specific staining).

Seventeen bladder transitional cell carcinomas of Grade III and StagesII or III and 12 normal bladder control samples were stained withantibody VB4-845 at 4 micrograms/ml (˜57 nM). Slide preparation andblocking were done according to well known immunohistochemistryprocedures. The detection of VB4-845 bound to tissue was done using arabbit anti-Pseudomonas exotoxin antibody (Sigma P2318), followed by abiotinylated anti-rabbit secondary antibody (Vector anti-rabbit BA-1000)and the Vector ABC-AP detection system using Vector red as substrate.

Carcinomas showed increased staining relative to normal transitionalepithelium, and the strongest staining observed in the positive caseswas membrane associated. Within carcinomas, the staining was variable inintensity and patchy in distribution. There was also an increasedstaining within areas exhibiting fair to moderate degrees ofdifferentiation (i.e. transitional or columnar differentiation) comparedto areas within the same tumor or tumors which showed lessdifferentiation or high degrees of nuclear anaplasia and pleomorphism.

Of the 17 transitional carcinomas stained, eight samples showed areas offaint to moderate membrane staining (2-3 on a 0-4 staining intensityscale (Samples 2, 6, 8, 11, 13, 15, and 16), one showed areas of faintstaining (Samples 9), and the other samples were negative for membranestaining. The staining was variable within the tumors, and appearedassociated with the degree of differentiation within the sample. Withinthe 12 normal bladder samples, two samples showed faint and lowfrequency membrane staining (Samples 2 and 11). No staining was seenwith a negative control immunotoxin (scFv-PE from an antibody to anirrelevant antigen).

Cytoplasmic and, more rarely, nuclear staining was seen in some normaland carcinoma specimens. In a validation study, higher concentration ofVB4-845 resulted in more “blush” or cytoplasmic staining but in a moreintense membrane staining on a higher percentage of carcinoma cells. Ina clinical setting (in vivo) since the cytoplasm and nucleus are notexposed to the product a higher concentration of VB4-845 could be usedto increase the binding to cells with lower number of receptors.

Example 15 Bladder Clinical Trial

In a clinical trial to evaluate the maximum tolerated dose of VB4-845,subjects with BCG-refractory transitional cell carcinoma (TCC) of thebladder, the drug is administered intravesically. The treatment cycleincludes 6 weeks of therapy and 4 to 6 weeks of follow-up. Theappropriate dose of VB4-845 will be administered via catheterizationdirectly into the bladder (tumor) once per week for 6 consecutive weeks.The 7 dose levels of VB4-845 are 100, 200, 335, 500, 700, 930, and 1240microgram in 50 ml at each of the 6 dosing day.

Immediately prior to drug administration, the bladder must be emptiedafter which a catheter will be inserted. For a male subject, a 16 FrenchCoude catheter with a Urojet will be used and for a female subject, a 14French red rubber catheter with sterile lubricant will be used.Reconstituted VB4-845 solution will be diluted in 50 ml of normalsaline, instilled into an empty bladder via catheterization, andretained in the bladder for 2 hours with the catheter clamped in place.At the end of 2 hours, the bladder will be emptied by unclamping thecatheter.

The safety, i.e. laboratory and adverse experience (AE) data at eachdose level will be evaluated after 3 weeks of treatment prior to doseescalation. The subject will continue weekly therapy at the determineddose level for a period of 6 weeks or until there is a dose limitingtoxicity (DLT) associated with the drug. Follow-up visits will beconducted within 4 to 6 weeks after the last week of drugadministration. A subject who experiences a DLT, but shows clinicalevidence of benefit to therapy will receive additional cycles oftreatment at the next lowest dose level once all toxicities haveresolved. Treatment will however be terminated for a subject whoexperiences a second DLT at the reduced dose. The response of the tumourwill be evaluated by cytology, cytoscopy and biopsy.

While the present invention has been described with reference to whatare presently considered to be the preferred examples, it is to beunderstood that the invention is not limited to the disclosed examples.To the contrary, the invention is intended to cover variousmodifications and equivalent arrangements included within the spirit andscope of the appended claims.

All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

TABLE 1 Sample VB4-845 Product Specifications Test Criteria AppearanceClear Solution at 2-8° C. Protein (BCA) 1.0 ± 0.2 mg/ml _(P)H 7.2 ± 0.2SDS-PAGE Major Band −70 kDa (Area ≧90%) (Non-Reducing: Coomassie Blue)Biological Activity (FACS) ≧50 fold increase in fluorescence over thecontrol antibody Cytotoxicity (IC₅₀) ≦0.50 pM Total DNA ≦1.0 ng/mgEndotoxin (LAL) ≦2000 EU/mg Sterility No Growth

TABLE 2 Summary of Effect of VB4-845 against Tumor Cells In Vitro Testsystem University Hospital of Zürich, Department of Information:Internal Medicine, Division of Medical Oncology, Zürich, SwitzerlandCell lines: SW2 small cell lung carcinoma CAL27 squamous cell carcinomaHT29 colorectal carcinoma COLO320 colorectal carcinoma MCF7 breastadenocarcinoma RL non-Hodgkin's lymphoma Dosage Form: VB4-845:0.0001-100 pM Assay: MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-disphenyltetrazolium bromide) assay Duration of Study: 72 hoursParameters Evaluated: Inhibition of cell growth by VB4-845 ObservedEffects and SW2, CAL27 and MCF7 cells were found Conclusions: to beequally sensitive to the cytotoxic effect of VB4-845 (IC₅₀ = 0.005 pM).HT29 cells were found to be the least sensitive (IC₅₀ of 0.2 pM).

TABLE 3 Summary Of Effect Of VB4-845 Agakst Protein Synthesis In TumorCells In Vitro Test system University Hospital of Zürich, Department ofInformation: Internal Medicine, Division of Medical Oncology, Zürich,Switzerland Cell lines: SW2 small cell lung carcinoma RL non-Hodgkin'slymphoma Dosage Form: VB4-845 - varying amounts Assay: Absorption of[4,5-³H]leucine Duration of Study: 30 hours Parameters Evaluated: Uptakeof [³H]leucine (measure of protein synthesis) Observed Effects andProtein synthesis was inhibited by VB4-845 Conclusions: in Ep-CAMpositive SW2 with an IC₅₀ of 0.01 pM. Protein synthesis in the Ep-CAMnegative control cell line RL was not affected

TABLE 4 Summary of Effect of VB4-845 on Solid Tumors in Mouse XenograftModels of Cancer Test Animal Mouse, athymic nude Information: UniversityHospital of Zürich, Department of Internal Medicine, Division of MedicalOncology, Zürich, Switzerland Animals implanted s.c. with one of: SW2small cell lung carcinoma CAL27 squamous cell carcinoma HT29 colorectalcarcinoma COLO320 colorectal carcinoma Dosage Form: VB4-845: 5 and 10 μg(see below) Route of Intravenous Administration: Treatment Regimen: i) 5μg every second day for 3 weeks (45 μg total) ii) 10 μg every second dayfor 1 week (30 μg total) Duration of Study: 50 days ParametersEvaluated: Primary tumor size Observed Effects and SW2: shrinkage of thetumor volume to maximal 20% of the initial Conclusions: size and aslight resumption of growth to a final 2.6-fold size increase at the endof the monitored period. CAL27: tumors reduced to maximal 60% of theinitial volume. The median tumor volume did not exceed 1.4-fold theinitial size 50 days post treatment initiation. Two mice out of 7treated with the 5 μg dose showed complete tumor regression and remainedtumor free. Neither CAL27 nor SW2 tumors showed a significant differencein their tumor response to the 2 treatment schedules. HT29: tumors sizedecreased 0.7-fold with 5 μg dose regimen. Three (3) out of 7 miceshowed complete regression of their HT29 tumors. The efficacy of the 10μg schedule was comparatively lower, indicating that for these tumors along-term treatment is more effective. No antitumor effect of VB4-845was seen in mice bearing Ep-CAM- negative COLO320 control tumors.

TABLE 5 Summary Of Effect Of Peritumoral Injection of VB4-845 On CAL27Squamous Cell Carcinoma Tumors In Mouse Xenograft Models Of Cancer TestAnimal Mouse, athymic nude Information: University Hospital of Zürich,Department of Internal Medicine, Division of Medical Oncology, Zürich,Switzerland Animals implanted s.c. with CAL27 squamous cell carcinomaDosage Form: VB4-845: 5 μg (see below) Route of PeritumoralAdministration: Treatment Regimen: 5 μg every second day (Mon/Wed/Fri)for 3 weeks (45 μg total) Duration of Study: 80 days ParametersEvaluated: Primary tumor size Observed Effects and Significantinhibition of tumor growth was Conclusions: observed in treated animals.Two mice showed complete tumor regression and remained tumor free forthe duration of the experiment.

TABLE 6 Summary of Effect of Escalating Repeat Doses of VB4-845 on TheLiver, Spleen and Bone of Immunocompetent Mice Test Animal Mouse,Immunocompetent C57BL/6 Information: University Hospital of Zürich,Department of Internal Medicine, Division of Medical Oncology, Zürich,Switzerland Dosage Form: VB4-845: 5 μg (see below) 10 μg 20 μg Route ofi.v Administration: Treatment Regimen: 5 or 10 μg every second day for 3doses (15 or 30 μg total, respectively) 20 μg every second day for 2doses (40 μg total) Study Groups: 3 animals/group 5 groups Duration ofStudy: 7 days Parameters Evaluated: Plasma ALT/AST Histopathologicalfindings, liver, spleen and bone Observed Effects and No elevation inliver enzymes 24 hours post final dose in the 5 or 10 μg Conclusions:dosing regimen mice. Elevated ALT/AST levels observed 24 hours postfinal dose in the 20 μg dose animals. No histopathological findings inthe 5 and 10 μg dose groups. A few sites with necrotic hepatocytes werefound in the 20 μg treatment group. No histopathological changes ormyelosuppresion observed in any dose group in the spleen or cellularcomponents of whole blood samples.

TABLE 7 Relationship Between Doses Used in Mouse Studies and TheProposed Low and Higher Dose of VB4-845 in Humans. Single Dose MonthlyOverall Exposure Multiple of Human Exposure Multiple of Human TotalExposure Multiple of Total (μg/kg) Dose (μg * kg) Monthly Dose (μg/kg)Human Dose Species Mouse (Low/High Dose)¹ Mouse (Low/High Dose)² Mouse(Low/High Dose)³ Athymic Mouse 250 862/63 2250  1585/113 2250 523/38Athymic Mouse 500 1724/125 1500 1056/75 1500 349/25 Athymic Mouse 250862/63 2250  1585/113 2250 523/38 C57BL/6 Mouse 250 862/63 750  528/38750 174/13 C57BL/6 Mouse 500 1724/125 1500 1056/75 1500 349/25 C57BL/6Mouse 1000 3448/250 2000  1408/100 2000 465/33 ¹0.29 and 4 μg/kg is theproposed low and higher single dose, respectively, for humanadministration (i.e., 20 μg and 280 μg administered to a 70 kgindividual). ²1.4 and 20 μg/kg is the proposed low and higher monthlydose, respectively, for human administration (i.e., 20 μg and 280 μgadministered to a 70 kg individual each day for five consecutive dayswith a three-week washout period). ³4.3 and 60 μg/kg is the proposed lowand higher total dose, respectively, for human administration (i.e., 20μg and 280 μg administered to a 70 kg individual each day for fiveconsecutive days with a three-week washout period for 3 cycles).

TABLE 8 Sample Carcinoma Carcinoma Positive Membrane % Cells numberGrade Stage Staining Positive 1 III II — — 2 III II Yes 40% 3 III II — —4 III II — — 5 III II — — 6 III II Yes 10% 7 III II — — 8 III II Yes 40%9 III III Yes 70% 10 III III — — 11 III III Yes 25% 12 III III — — 13III III Yes 30-40% 14 III III — — 15 III III Yes 10% 16 III III Yes 30%17 III III — —

TABLE 9 Characteristics of Bladder Cancer Cell Lines Ref. Bladder CancerPrimary Tumour Tumour no. Cell Lines Tissue of Origin Grade Tumour StageDifferentiation 1 1A6 Bladder TCC High Invasive Well 2 T-24 Bladder TCCHigh Invasive Poor 3 SW-780 Bladder TCC Low Invasive No data 4 HT-1197Bladder TCC High Invasive Poor 5 RT-4 Bladder TCC Low Superficial(non-invas.) Well 6 SCaBER Bladder SqCC No data Invasive Moderately 7HT-1376 Bladder TCC High Invasive Poor 8 TCCSUP Bladder TCC HighInvasive Poor 9 J-82 Bladder SqCC High Invasive Poor 10 UM-UC-3 BladderTCC High Invasive Poor 10 UM-UC-13 Bladder TCC High Invasive No data 11UM-UC-10 No data No data No data No data 11 UM-UC-14 No data No data Nodata No data 1 5636 Bladder TCC High Invasive No data References: 1: Aclone of the parent cell line, 5637, Immunobiol. 172: 175-184 (1986),Urol. Res. 21: 27-32 (1993); 2: Int. J. Cancer 11: 765-773 (1973), J.Urol. 149: 1626-1632 (1993); 3: Cancer Res. 44: 3997-4005 (1984); 4: J.Natl. Cancer Inst. 58: 881-890 (1977); 5: J. Urol. 161: 692-698 (1999);6: Int. J. Cancer 17; 707-714 (1976); 7: J. Natl. Cancer Inst. 58:881-890 (1977); 8: Br. J. Cancer 35: 142-151 (1977); 9: Br. J. Cancer38: 64-76 (1978); 10: J. Urol. 146: 227-231 (1991); 11: AntiCancer Inc.,Cell lines. TCC: Transitional cell carcinoma. SqCC: Squamous cellcarcinoma.

TABLE 10 Tumor Cell-Surface Reactivity of VB4-845 Bladder CancerReactivity¹: Cytotoxicity Cytotoxicity: Relative Cell LinesFold-Increase in Fluorescence IC₅₀ (pM) Sensitivity vs. CAL27² 1A6 154.7± 15.2 0.033 ± 0.01 8.8 T-24 134.1 ± 35.9 0.001 ± 0.0  290 UM-UC-10124.6 ± 5.3 0.024 ± 0.00 12.1 5637  97.0 ± 11.2  0.38 ± 0.13 0.8 SW-780 86.7 ± 3.1 0.002 ± 0.00 145 HT-1197  56.5 ± 2.3  0.23 ± 0.05 1.3 RT-4 55.3 ± 16.4  0.20 ± 0.10 1.4 SCaBER  54.0 ± 2.1 10.1 ± 0.0 0.03 HT-1376 40.7 ± 0.3  3.3 ± 1.2 0.1 UM-UC-14  25.7 ± 1.2 0.17 ± 0.2 1.6 TCCSUP 2.0 ± 0.1  320.0 ± 102.0 0.0009 J-82  1.2 ± 0.1² >500 n/a UM-UC-3  1.2± 0.1² >500 n/a UM-UC-13  1.3 ± 0.1² >500 n/a CAL-27  87.0 ± 3.0 0.29 ±0.1 1.0 (Positive control) COLO-320  1.1 ± 0.1² >500 n/a (Negativecontrol) ¹Fold-increase in median fluorescence above the control. Thevalues are expressed as mean ± SEM. The reactivity of the antibody for agiven indication was determined by averaging mean-fold increase inmedian fluorescence calculated for each cell line in that indication.²Cell lines showing a positive shift in fluorescence of <30% (1.3-foldincrease) were considered negative.

REFERENCES

-   1. Chaubai S, Wollenberg B, Kastenbauer E, Zeidler R (1999) Ep-CAM—a    marker for the detection of disseminated tumor cells in patients    suffering from SCCHN. Anticancer Res JID—8102988 19:2237-2242-   2. Salter E R, Tichansky D, Furth E E, Herlyn A M (2001)    Tumor-associated antigen expression and growth requirements predict    tumorigenesis in squamous cell carcinoma. In Vitro Cell Dev Biol    Anim JID—9418515 37:530-535-   3. Takes R P, Baatenburg dJ R, Schuuring E, Litvinov S V, Hermans J,    van Krieken J H (1998) Differences in expression of oncogenes and    tumor suppressor genes in different sites of head and neck squamous    cell. Anticancer Res JID—8102988 18:4793-4800-   4. Oppenheimer N J, Bodley J W (1981) Diphtheria toxin. Site and    configuration of ADP-ribosylation of diphthamide in elongation    factor 2. J Biol Chem JID—2985121R 256:8579-8581-   5. Kreitman R J (1999) Immunotoxins in cancer therapy. Curr Opin    Immunol 11:570-578-   6. Kreitman R J (2000) Immunotoxins. Expert Opin Pharmacother    1:1117-1129-   7. Grossbard M L, Nadler L M (1993) Monoclonal antibody therapy for    indolent lymphomas. Semin Oncol 20:118-135-   8. Wahl R L (1994) Experimental radioimmunotherapy. A brief    overview. Cancer 73:989-992-   9. Grossbard M L, Fidias P (1995) Prospects for immunotoxin therapy    of non-Hodgkin's lymphoma. Clin Immunol Immunopathol 76:107-114-   10. Jurcic J G, Caron P C, Scheinberg D A (1995) Monoclonal antibody    therapy of leukemia and lymphoma. Adv Pharmacol 33:287-314-   11. Lewis J P, DeNardo G L, DeNardo S J (1995) Radioimmunotherapy of    lymphoma: a UC Davis experience. Hybridoma 14:115-120-   12. Uckun F M, Reaman G H (1995) Immunotoxins for treatment of    leukemia and lymphoma. Leuk Lymphoma 18:195-201-   13. Kreitman R J, Wilson W H, Bergeron K, Raggio M,    Stetler-Stevenson M, FitzGerald D J, Pastan I (2001) Efficacy of the    anti-CD22 recombinant immunotoxin BL22 in chemotherapy-resistant    hairy-cell leukemia. N Engl J Med 345:241-247-   14. Schwartzberg L S (2001) Clinical experience with edrecolomab: a    monoclonal antibody therapy for colorectal carcinoma. Crit. Rev    Oncol Hematol JID—8916049 40:17-24-   15. Adkins J C, Spencer C M (1998) Edrecolomab (monoclonal antibody    17-1 A). Drugs JID—7600076 56:619-626-   16. Litvinov S V, Velders M P, Bakker H A, Fleuren G J, Warnaar S    O (1994) Ep-CAM: a human epithelial antigen is a homophilic    cell-cell adhesion molecule. J Cell Biol JID—0375356 125:437-446-   17. Willuda J, Honegger A, Waibel R, Schubiger P A, Stahel R,    Zangemeister-Wittke U, Pluckthun A (1999) High thermal stability is    essential for tumor targeting of antibody fragments: engineering of    a humanized anti-epithelial glycoprotein-2 (epithelial cell adhesion    molecule) single-chain Fv fragment. Cancer Res JID—2984705R    59:5758-5767-   18. Proca D M, Niemann T H, Porcell A I, DeYoung B R (2000) MOC31    immunoreactivity in primary and metastatic carcinoma of the liver.    Report of findings and review of other utilized markers. Appl    Immunohistochem Mol Morphol JID—100888796 8:120-125-   19. Pavlovskis O R, Gordon F B (1972) Pseudomonas aeruginosa    exotoxin: effect on cell cultures. J Infect Dis JID—0413675    125:631-636-   20. Leppla S H (1976) Large-scale purification and characterization    of the exotoxin of Pseudomonas aeruginosa. Infect Immun JID—0246127    14:1077-1086-   21. Kreitman R J, Pastan I (1998) Accumulation of a recombinant    immunotoxin in a tumor in vivo: fewer than 1000 molecules per cell    are sufficient for complete responses. Cancer Res JID—2984705R    58:968-975-   22. Perentesis J P, Miller S P, Bodley J W (1992) Protein toxin    inhibitors of protein synthesis. Biofactors JID—8807441 3:173-184-   23. Milenic D E, Yokota T, Filpula D R, Finkelman M A, Dodd S W,    Wood J F, Whitlow M, Snoy P, Schlom J (1991) Construction, binding    properties, metabolism, and tumor targeting of a single-chain Fv    derived from the pancarcinoma monoclonal antibody CC49. Cancer Res.    51:6363-6371-   24. Yokota T, Milenic D E, Whitlow M, Schlom J (1992) Rapid tumor    penetration of a single-chain Fv and comparison with other    immunoglobulin forms. Cancer Res. 52:3402-3408-   25. Verhaar M J, Keep P A, Hawkins R E, Robson L, Casey J L, Pedley    B, Boden J A, Begent R H, Chester K A (1996) Technetium-99m    radiolabeling using a phage-derived single-chain Fv with a    C-terminal cysteine. J. Nucl. Med. 37:868-872-   26. Adams G P, McCartney J E, Tai M S, Oppermann H, Huston J S,    Stafford W F, Bookman M A, Fand I, Houston L L, Weiner L M (1993)    Highly specific in vivo tumor targeting by monovalent and divalent    forms of 741F8 anti-c-erbB-2 single-chain Fv. Cancer Res.    53:4026-4034-   27. Deonarain M P, Rowlinson-Busza G, George A J, Epenetos A    A (1997) Redesigned anti-human, placental alkaline phosphatase    single-chain Fv: soluble expression, characterization and in vivo    tumour targeting. Protein Eng. 10:89-98-   28. Friedman P N, McAndrew S J, Gawlak S L, Chace D, Trail P A,    Brown J P, Siegall C B (1992) BR96 sFv-PE40, a potent single-chain    immunotoxin that selectively kills carcinoma cells. Cancer Res.    53:334-339-   29. Begent R H, Verhaar M J, Chester K A, Casey J L, Green A J,    Napier M P, Hope-Stone L D, Cushen N, Keep P A, Johnson C J, Hawkins    R E, Hilson A J, Robson L (1996) Clinical evidence of efficient    tumor targeting based on single-chain Fv antibody selected from a    combinatorial library. NatMed. 2:979-984-   30. Mayer A, Tsiompanou E, O'Malley D, Boxer G M, Bhatia J, Flynn    A A. Chester K A, Davidson B R, Lewis A A, Winslet M C, Dhillon A P,    Hilson A J, Begent R H (2000) Radioimmunoguided surgery in    colorectal cancer using a genetically engineered anti-CEA    single-chain Fv antibody. Clin Cancer Res 6:1711-1719-   31. Kreitman R J, Wilson W H, Robbins D, Margulies I,    Stetler-Stevenson M, Waldmann T A, Pastan I (1999) Responses in    refractory harry cell leukemia to a recombinant immunotoxin. Blood    94:3340-3348-   32. Kreitman R J, Wilson W H, White J D, Stetler-Stevenson M, Jaffe    E S, Giardina S, Waldmann T A, Pastan I (2000) Phase I trial of    recombinant immunotoxin anti-Tac(Fv)-PE38 (LMB-2) in patients with    hematologic malignancies. J Clin Oncol 18:1622-1636-   33, Bodey B, Siegel S E, Kaiser H E (1996) Human cancer detection    and immunotherapy with conjugated and non-conjugated monoclonal    antibodies. Anticancer Res. 16:661-674-   34. Multani P S, Grossbard M L (1998) Monoclonal antibody-based    therapies for hematologic malignancies. J. Clin. Oncol. 16:3691-3710-   35. White C A, Larocca A, Grillo-Lopez A J (1999) Anti-CD20    monoclonal antibodies as novel treatments for non-Hodgkin's    lymphoma. PSTT 2:95-101-   36. Saleh M N, Posey J A, Khazaeli M B, Thurmond L M, Khor S P,    Lampkin T A, Wissel P S, LoBuglio A F (1998) Phase I trial testing    multiple doses of humanized monoclonal antibody (MAb) 3622W94. ASCO    1998 meeting #1680 (Abstract)-   37. Raum T, Gruber R, RiethmuUer G, Kufer P (2001) Anti-self    antibodies selected from a human IgD heavy chain repertoire: a novel    approach, to generate therapeutic human antibodies against    tumor-associated differentiation antigens. Cancer Immunol Immunother    JED—8605732 50:141-150-   38. Kroesen B J, Nieken J, Sleijfer D T, Molema G, de Vries E G,    Grcen H J, Helfrich W, The T H, Mulder N H, de Leij L (1997)    Approaches to lung cancer treatment using the CD3×EGP-2-directed    bispecific monoclonal antibody BIS-1. Cancer Immunol Immunother    JID—8605732 45:203-206-   39. Haller D G (2001) Update of clinical trials with edrecolomab: a    monoclonal antibody therapy for colorectal cancer. Semin Oncol    28:25-30-   40. Riethmuller G, Holz E, Schlimok G, Schmiegel W, Raab R, Hoffken    K, Gruber R, Funke I, Pichlmaier H, Hirche H, Buggisch P, Witte J,    Pichlmayr R (1998) Monoclonal antibody therapy for resected Dukes' C    colorectal cancer: seven-year outcome of a multicenter randomized    trial, J Clin Oncol JID—8309333 16:1788-1794-   41. Dencausse Y, Hartung G, Franz A, Strum J, Edler L, Bornbusch D,    Gonnermann M, Post S, Hehlmann R, Queisser W (2000) Prospective    randomized study of adjuvant therapy with edrecolomab (PANOREX) of    stage II colon cancer: Interum analysis. Ann. Oncol. 11:47(Abstract)-   42. de Boer C J, van Krieken J H, Janssen-van Rhijn C M, Litvinov S    V (1999) Expression of Ep-CAM in normal, regenerating, metaplastic,    and neoplastic liver. J Pathol JID—0204634 188:201-206-   43. Balzar M, Winter M J, de Boer C J, Litvinov S V (1999) The    biology of the 17-1A antigen (Ep-CAM). J Mol Med JID—9504370    77:699-712-   44. Herlyn D, Sears H F, Ernst C S, Iliopoulos D, Steplewski Z,    Koprowski H (1991) Initial clinical evaluation of two murine IgG2a    monoclonal antibodies for immunotherapy of gastrointestinal    carcinoma. Am J Clin Oncol JID—8207754 14:371-378-   45. Begent R H, Chester K A (1997) Single-chain Fv antibodies for    targeting cancer therapy. Biochem. Soc. Trans. 25:715-717-   46. Chester K A, Mayer A, Bhatia J, Robson L, Spencer D I, Cooke S    P, Flynn A A, Sharma S K, Boxer G, Pedley R B, Begent R H (2000)    Recombinant anti-carcinoembryonic antigen antibodies for targeting    cancer. Cancer Chemother Pharmacol 46 Suppl:S8-12-   47. Chester K A, Bhatia J, Boxer G, Cooke S P, Flynn A A, Huhalov A,    Mayer A, Pedley R B, Robson L, Sharma S K, Spencer D I, Begent R    H (2000) Clinical applications of phage-derived sFvs and sFv fusion    proteins. Dis Markers 16:53-62-   48. Grossbard M L, Freedman A S, Ritz J, Coral F, Goldmacher V S,    Eliseo L, Spector N, Dear K, Lambert J M, Blattler W A (1992)    Serotherapy of B-cell neoplasms with anti-B4-blocked ricin: a phase    I trial of daily bolus infusion. Blood 79:576-585-   49. Amlot P L, Stone M J, Cunningham D, Fay J, Newman J, Collins R,    May R, McCarthy M, Richardson J, Ghetie V (1993) A phase I study of    an anti-CD22-deglycosylated ricin A chain immunotoxin in the    treatment of B-cell lymphomas resistant to conventional therapy.    Blood 82:2624-2633-   50. Vitetta E S, Stone M, Amlot P, Fay J, May R, Till M, Newman J,    Clark P, Collins R, Cunningham D (1991) Phase I immunotoxin trial in    patients with B-cell lymphoma. Cancer Res 51:4052-4058-   51. Stone M J, Sausville E A, Fay J W, Headlee D, Collins R H, Figg    W D, Stetler-Stevenson M, Jain V, Jaffe E S, Solomon D, Lush R M,    Senderowicz A, Ghetie V, Schindler J, Uhr J W, Vitetta E S (1996) A    phase I study of bolus versus continuous infusion of the anti-CD19    immunotoxin, IgG-HD37-dgA, in patients with B-cell lymphoma. Blood    JID—7603509 88:1188-1197-   52. Messmann R A, Vitetta E S, Headlee D, Senderowicz A M, Figg W D,    Schindler J, Michiel D F, Creekmore S, Steinberg S M, Kohler D,    Jaffe E S, Stetler-Stevenson M, Chen H, Ghetie V, Sausville E    A (2000) A phase I study of combination therapy with immunotoxins    IgG-HD37-deglycosylated ricin A chain (dgA) and IgG-RFB4-dgA    (Combotox) in patients with refractory CD19(+), CD22(+) B cell    lymphoma. Clin Cancer Res JID—9502500 6:1302-1313-   53. Di Paolo, C, Willuda, J., Kubetzko, S., Lauffer, I., Tschudi,    D., Waibel, R., Pluckthun, A., Stahel, R. A., and    Zangemeister-Witte, U. A recombinant immunotoxin derived from a    humanized Ep-CAM-specific single-chain antibody fragment has potent    and selective antitumor activity, (submitted)-   54. Di Paolo, C. and Zangemeister-Witte, U., Personal communication,    Zurich.-   55. Schumann, J., Angermuller, S., Bang, R., Lohoff, M., and    Tiegs, G. Acute hepatotoxiciry of Pseudomonas aeruginosa exotoxin A    in mice depends on T cells and TNF. J. Immunol, 161: 5745-5754,    1998.-   56. Schümann, J., Wolf, D., Pahl, A., Brune, K., Papadopoulos, T.,    van Rooijen, N., and Tiegs, G. Importance of Kupffer cells for    T-cell-dependent liver injury in mice. Am. J. Pathol, 157:    1671-1683, 2000.-   57. Sizmann N and Korting H C, Prolonged Urticaria with 17-1A    Antibody. BMJ 317:1631. F Fichtner I, Kufer P, Raum T, Riethmuller    G, Baeuerle P A, Dreier T. In vitro and in vivo activity of MT201, a    fully human monoclonal antibody for pancarcinoma treatment. Int J    Cancer 100(i): 101-10, 2002.-   59. Willuda J, Honegger A, Waibel R, Schubiger P A, Stahel R,    Zangemeister-Wittke U, Pluekthun A. High thermal stability is    essential for tumor targeting of antibody fragments: engineering of    a humanized anti-epithelial glycoprotein-2 (epithelial cell adhesion    molecule) single-chain Fv fragment. Cancer Res 59(22):5758-67, 1999-   60. Wels, W., Beerli, R., Hellmann, P., Schmidt, M., Marte, B. M.,    Kornilova, E. S., Hekele, A., Mendelsohn, J., Groner, B., and    Hynes, N. E. EGF receptor and p185erbB-2-specific single-chain    antibody toxins differ in their cell killing activity on tumor cells    expressing both receptor proteins. Int. J. Cancer, 60: 137-144,    1995.-   61. Ge, L., Plückthun, A., Pack, P., Freund, C, and Pluckthun, A.    Expressing antibodies in Escherichia coli. In C. A. K. Borrebaeck    (ed.), Antibody engineering, pp. 229-261. Oxford: Oxford University    Press, 1995.-   62. Willuda, J., Honegger, A., Waibel, R., Schubiger, P. A., Stahel,    R., Zangemeister-Wittke, U., and Pliickthun, A. High thermal    stability is essential for tumor targeting of antibody fragments:    engineering of a humanized anti-epithelial glycoprotein-2    (epithelial cell adhesion molecule) single-chain Fv fragment. Cancer    Res., 59: 5758-5767, 1999.-   63. Bass, S., Gu, Q., and Christen, A. Multicopy suppressors of prc    mutant Escherichia coli include two HtrA (DegP) protease homologs    (HhoAB), DksA, and a truncated RlpA. J. Bacteriol, 178: 1154-1161,    1996.-   64. Pluckthun, A., Krebber, A., Krebber, C, Horn, U., Kntipfer, U.,    Wenderoth, R., Nieba, L., Proba, K., and Riesenberg, D. Producing    antibodies in Escherichia Coli: from PCR to fermentation. In J.    McCafferty, H. R. Hoogenboom, and D. J. Chiswell (eds.), Antibody    engineering, pp. 203-252. Oxford: IRL Press, 1996.-   65. Waibel, R., Alberto, R., Willuda, J., Finnern, R., Schibli, R.,    Stichelberger, A., Egli, A., Abram, U., Mach, J. P., Plückthun, A.,    and Schubiger, P. A. Stable one-step technetium-99m labeling of    His-tagged recombinant proteins with a novel Tc(I)-carbonyl complex.    Nat. Biotechnol., 17: 897-901, 1999.-   66. Lindmo, T., Boven, E., Cuttitta, F., Fedorko, J., and Bunn, P.    A., Jr. Determination of the immunoreactive fraction of radiolabeled    monoclonal antibodies by linear extrapolation to binding at infinite    antigen excess. J. Immunol. Methods, 72: 77-89, 1984.

ABBREVIATIONS

ADME: administration, distribution, metabolism and excretion

ADP: adenosine phosphate

ALT: alanine aminotransferase (SGPT)

AST: aspartate aminotransferase (SGOT)

BCA: bieinchoninic acid method

C_(max): maximum concentration

DNA: deoxyribonucleic acid

Ep-CAM: epithelial cell adhesion molecule

ETA: Pseudomonas exotoxin A

EU: endotoxin units

FACS: fluorescence activated cell sorter method

GLP: good laboratory practices

HNSCC: squamous cell carcinoma of the head and neck

IC₅₀: inhibitory concentration 50%

i.t. intratumoral

i.v. intravenous

kDa: kilodalton

LAL: Limulus amebocyte lysate

MAbs: monoclonal antibodies

mg: milligram

mL: milliliter

mM: millimolar

MTD: maximum tolerated dose

MTT: 3-[4,5-dimethylthiazol-2-yl]-2,5-disphenyltetrazolium

NaCl: Sodium chloride

ng: nanogram

PBS: phosphate buffered saline

pi: isoelectric point

PK: pharmacokinetics

pM: picomolar

p.t: peritumoral

s.c: subcutaneous

scFv; single chain antibody fragment

SCLC: small cell lung cancer

SD: standard deviation

SDS PAGE: sodium dodesyl sulfate polyacrylamide gel electrophoresis

t_(1/2) half-life

T_(max) time to maximum

μg microgram

VLS: vascular leak syndrome

WHO: World Health Organization

wt: wild type

The present invention is not to be limited in scope by the specificembodiments described above. Many modifications of the presentinvention, in addition to those specifically recited above would beapparent to those skilled in the art in light of the instant disclosure.These modifications are intended to fall within the scope of theappended claims. All publications and patents cited above are hereinincorporated by reference in their entireties.

What is claimed is:
 1. An immunotoxin comprising from amino acid 23 toamino acid 669 of SEQ ID NO:2.
 2. The immunotoxin of claim 1, whereinthe immunotoxin comprises the amino acid sequence of SEQ ID NO:
 2. 3. Amethod of treating bladder cancer comprising administering, directly tothe cancer site, an effective amount of an immunotoxin to an animal inneed thereof, wherein said immunotoxin comprises the amino acid sequencefrom amino acid 23 to amino acid 669 of SEQ ID NO:
 2. 4. The method ofclaim 3, wherein the immunotoxin is VB4-845 consisting of the amino acidsequence from amino acid 23 to amino acid 669 of SEQ ID NO:
 2. 5. Amethod for treating bladder cancer comprising: testing a tumor samplefrom a patient for the expression of a protein suspected of beingassociated with bladder cancer wherein the protein is Epithelial CellAdhesion Molecule (Ep-CAM); and if the protein is expressed at greaterlevels in the tumor sample as compared to a control, administering tothe patient, directly to the cancer site, an effective amount ofimmunotoxin, comprising the amino acid sequence from amino acid 23 toamino acid 669 of SEQ ID NO:
 2. 6. The method of claim 3 wherein theanimal in need thereof is a human.
 7. The method of claim 3 wherein theimmunotoxin is administered to the animal before, during or aftersurgery.
 8. The method of claim 3 wherein the immunotoxin isadministered to the animal before, during or after radiation therapy. 9.The method of claim 3 wherein the immunotoxin is administered to theanimal before, during or after one or more chemotherapeutic agents. 10.The method of claim 9 wherein the one or more chemotherapeutic agent isone or more of cisplatin, fluorouracil, carboplatin, mitomycin C,doxorubicin, gemcitabine, or paclitaxel.
 11. The method of claim 10wherein the one or more chemotherapeutic agent is flurouracil.
 12. Themethod of claim 10 wherein the one or more chemotherapeutic agent is oneor more of mitomycin C, doxorubicin, or gemcitabine.
 13. The method ofclaim 3 wherein the immunotoxin is administered once per week for 6weeks.
 14. The method of claim 3 wherein the immunotoxin is administeredto an animal in combination with one or more agents that increasesexpression of EpCAM in the tumor cells.
 15. The method of claim 14wherein the one or more agents that increases expression of EpCAM in thetumor cells is vinorelbine tartrate and/or paclitaxel.
 16. The method ofclaim 3 wherein the immunotoxin is administered with a catheter directlyinto the bladder of said animal.
 17. The method of claim 3 wherein thebladder cancer is BCG refractory transitional carcinoma.
 18. The methodof claim 3 wherein the treating comprises decreasing the size, growthrate, invasiveness, malignancy grade and/or risk of recurrence of atumor associated with the bladder cancer.